A method for directly preparing a high-viscosity fermentation emulsion by biological fermentation and a product thereof

Abstract

The present disclosure provides a method for directly preparing high-viscosity fermentation emulsion by biological fermentation, comprising the following steps: step one, preparing white ginseng mycelium by liquid fermentation culture; step two, proportionally configuring the white ginseng mycelium and water into a substrate, sterilizing, inoculating white ginseng seed liquid, performing fermentation culture, centrifuging to obtain supernatant, sterilizing the supernatant to obtain fermentation liquid. The present disclosure also provides a high-viscosity fermentation emulsion prepared by the above method. The high-viscosity fermentation emulsion prepared by the method of directly fermenting mycelium by biology improves the viscosity of the fermentation liquid, improves the skin feel, and has good soothing, anti-wrinkle and moisturizing properties.

Description

TECHNICAL FIELD

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

[0002] Generally, emulsion is obtained by formula design, emulsification of water phase and oil phase by the action of emulsifier, and finally by emulsification operation. The emulsion has high water phase content, good fluidity, small viscosity, and is difficult to maintain stability, and is prone to delamination during storage. In the formula design and preparation of the emulsion, a thickening agent such as a water-soluble gum raw material or a water-soluble polymer compound is often added to make the density of the dispersed phase and the dispersion medium as close as possible to obtain a stable emulsion system. The selection of emulsifier and emulsification equipment is required to be higher.

[0003] The present application directly uses oil and fat as a fermentation substrate before fermentation by a biological fermentation method, and high-viscosity fermentation milk is obtained by the fermentation of microorganisms and direct separation at the end of fermentation. The method of the present application does not need to add an emulsifier, and there is no formula design and emulsification process in the later stage, and the fermentation milk obtained by pure biological manufacturing is white to light yellow in color, has a moisturizing skin feel, is good in absorption, and is natural. SUMMARY

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

[0005] 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 high-viscosity fermentation emulsion by biological fermentation, comprising:

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

[0008] Step two, configuring the white ginseng fungus mycelium and water into a substrate in proportion, sterilizing, inoculating white ginseng fungus seed liquid, and performing fermentation culture, centrifuging to obtain supernatant, sterilizing to obtain fermentation liquid.

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

[0010] In the above preparation method, as a preferred embodiment, in step one, the preparation method of the white ginseng fungus mycelium is as follows: adding 2% of potatoes and 2% of sucrose into water, then performing homogenization treatment, inoculating white ginseng fungus seed liquid for fermentation culture, and finally separating to obtain the white ginseng fungus mycelium. In specific examples, for example, 6 g of potatoes and 6 g of sucrose can be added into 300 mL of water, then homogenization treatment is performed, then white ginseng fungus seed liquid is inoculated for fermentation culture, and finally the white ginseng fungus mycelium is separated.

[0011] In the above preparation method, as a preferred embodiment, in step one and / or step two, the volume ratio of the white ginseng fungus mycelium in the white ginseng fungus seed liquid is 75-90% (such as 78%, 80%, 82%, 85%, 88%, etc.), preferably 80-90% of the entire liquid culture medium; the inoculation ratio, i.e., 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 ratio of the white ginseng fungus mycelium in the white ginseng fungus seed liquid is 85-90% of the entire liquid culture medium, and the preparation method of the white ginseng fungus seed liquid comprises the following steps: (1) taking 2-3 solid strains with a diameter of about 0.5 cm from the white ginseng fungus solid strain growing in a PDA plate together with the culture medium, inoculating into 300 mL of potato dextrose water culture medium, and culturing at 28°C under 180 rpm for 3 days to obtain white ginseng fungus seed liquid 1, at this time the white ginseng fungus seed liquid 1 has different sizes of mycelial pellets; (2) homogenizing the white ginseng fungus seed liquid 1 and taking 10 mL as seed liquid to inoculate into 300 mL of potato dextrose water culture medium, and culturing at 28°C under 180 rpm for 3 days to obtain white ginseng fungus seed liquid 2, at this time the volume ratio of the white ginseng fungus mycelium is about 85-90% of the entire liquid culture medium.

[0012] In the above preparation method, as a preferred embodiment, in step two, the mass ratio of the white ginseng fungus mycelium to water in the substrate is 1:5-1:15 (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, in step two, sucrose is further included, and the addition amount of sucrose 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 two, mung bean starch is further included, and the addition amount of mung bean starch is 0-3% (such as 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, etc.), preferably 0.5%; further, macadamia seed oil is further included, and the addition amount of macadamia seed oil is 0-3% (such as 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), homogenization treatment is directly performed, and then the treatment in step two is performed as a substrate; more preferably, in step two, the substrate further includes oil, and the oil is selected from at least one of white pool flower seed oil, soybean oil, flaxseed oil, and corn germ oil, and the addition 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.). Further, the fermentation culture is performed 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 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 disclosure include but are not limited to:

[0019] 1. The present application directly improves the viscosity of the fermentation broth by the method of biological fermentation mycelium, and improves the skin feel;

[0020] 2. In the present application, oil is directly used as a substrate before fermentation by the method of biological fermentation, and through the fermentation effect of microorganisms, a high-viscosity fermented emulsion can be obtained by direct separation after fermentation. The method of the present application does not need to add an emulsifier, and there is no formula design and emulsification process in the later stage, and it is purely biological manufacturing. The fermented milk obtained by the method of the present application is white to light yellow in color, has a moisturizing skin feel, is easy to absorb, and is natural.

[0021] 3. The high-viscosity fermented emulsion prepared by the present application has good soothing, anti-wrinkle, and moisturizing properties.

[0022] 4. The preferred preparation method of this application is in-situ fermentation, that is, the stage of obtaining mycelium is the first fermentation step. Instead of collecting mycelium separately, the mycelium and fermentation broth from the first fermentation step are directly processed together to carry out the second fermentation step. This makes it easier to achieve large-scale production, omits the mycelium separation and preservation steps, and saves production energy. Attached Figure Description

[0023] Figure 1 Photographs of *Gynostemma pentaphyllum* seed solutions (YSC-1 seed solution, 290 seed solution);

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

[0025] Figure 3 Figures (a) and (b) show the DPPH free radical scavenging capacity curves of the lyophilized fermentation supernatant obtained in Example 6 and Comparative Example 2.1, respectively.

[0026] Figure 4 The sensory evaluation comparison chart shows the fermentation broths prepared in Example 6 and Comparative Example 2.1.

[0027] Figure 5 The sensory evaluation comparison chart shows the fermentation liquid prepared in Example 6 and Comparative Example 2.1 and a commercially available face mask (natural fermented face mask).

[0028] Figure 6 Sensory evaluation comparison chart of emulsions made from the fermentation broths obtained in Examples 7 and 8;

[0029] Figure 7 Sensory evaluation comparison chart of emulsions made from the fermentation broths obtained in Examples 7 and 8 and commercially available emulsions;

[0030] Figure 8 The anti-wrinkle performance test results of the mask liquid prepared in Example 6 and Comparative Example 2.1, and the emulsion prepared in Example 7 and Example 8 are shown.

[0031] Figure 9 The results of the moisturizing performance tests of the mask liquid prepared in Example 6 and Comparative Example 2.1, and the emulsion prepared in Example 7 and Example 8 are shown.

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

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

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

[0035] Figure 13 The images show the sample photos obtained in Examples 9-14 and Comparative Examples 6-8, respectively. Detailed Implementation

[0036] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0037] The technical solutions of this disclosure will be described below with reference to exemplary embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

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

[0039] The preparation method of the *Bletilla striata* seed liquid (also known as 290 seed liquid, YSC-1 seed liquid) used in the examples / comparative examples includes the following steps: (1) Inoculate 2-3 solid *Bletilla striata* YSC-1 culture medium (with culture medium, using a sterilized punch to take 2-3 solid culture medium with a diameter of about 0.5 cm) grown on a PDA plate into 300 mL of potato dextrose water culture medium, and culture at 28°C and 180 rpm for 3 days to obtain *Bletilla striata* seed liquid 1. At this time, the mycelial balls in *Bletilla striata* seed liquid 1 are of different sizes; (2) Homogenize the *Bletilla striata* seed liquid 1 obtained in step (1) using a sterile glass homogenizer, and take 10 mL as seed liquid to inoculate into 300 mL of potato dextrose water culture medium, and culture at 28°C and 180 rpm for 3 days to obtain *Bletilla striata* seed liquid 2. At this time, the YSC-1 mycelial balls are suspended in the fermentation broth. See Figure 1The proportion of the volume of the mycelium of the white ginseng fungus in the entire liquid culture medium was about 85-90%, and this was used as the seed liquid of the white ginseng fungus in the preparation of samples in subsequent experiments, unless otherwise specified.

[0040] The white pool flower seed oil used in the examples / counter-examples was purchased from Qingdao Biyouti Chemical Technology Co., Ltd., the macadamia nut seed oil was purchased from Heye International Trade (Shanghai) Co., Ltd., the flaxseed oil, soybean oil, and corn germ oil were all purchased from Jingdong; the potato dextrose aqueous culture medium used was purchased from Haibo Biotechnology Co., Ltd. in Qingdao High-tech Industrial Park (product number: HB0233-4). The potatoes used in the examples / counter-examples were all fresh potatoes.

[0041] The viscosity measurement used a DV2T viscometer, Brookfield Ametek, parameters: No. 2 rotor 60 rpm. The solid content measurement used an LG18T solid concentration meter, Guangzhou Mingrui Electronics Technology Co., Ltd.

[0042] I. Examples 1-5 and counter-examples 1.1-3

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

[0044] Example 1

[0045] (1) 300 mL of potato dextrose aqueous culture medium was inoculated with 10 mL of white ginseng fungus seed liquid (also referred to as 290 seed liquid in this application, YSC-1 seed liquid), the temperature was 28°C, the rotation speed was 180 rpm, and the mycelium was collected by centrifugation after 3 days of culture.

[0046] (2) Fresh mycelium and water were homogenized at a ratio of 1:10 (mass ratio), and then 300 mL of each was dispensed into a bottle, 10 mL of YSC-1 seed liquid was inoculated after sterilization at 121°C for 15 min, the temperature was 28°C, the rotation speed was 180 rpm, and the fermentation supernatant (i.e. fermentation broth) was obtained by centrifugation after 3 days of culture. The viscosity of the supernatant was 302 Pa·s, the solid content (referred to as "solid content" for short) was 1.30%, and the pH was 6.31.

[0047] Example 2

[0048] (1) The mycelium was prepared as in example 1.

[0049] (2) Fresh mycelium and water were homogenized at a ratio of 1:10 (mass ratio), 300 mL per bottle, 0.5% sucrose (1.5 g) was added according to the ratio, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, cultured at 28°C for 3 days at 180 rpm, centrifuged to obtain fermentation supernatant and measure viscosity, solid content and pH.

[0050] Example 3

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

[0052] (2) Fresh mycelium and water were homogenized at a ratio of 1:10 (mass ratio), 300 mL per bottle, 0.5% mung bean starch (1.5 g) was added according to the ratio, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, cultured at 28°C for 3 days at 180 rpm, centrifuged to obtain fermentation supernatant and measure viscosity, solid content and pH.

[0053] Example 4

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

[0055] (2) Fresh mycelium and water were homogenized at a ratio of 1:10 (mass ratio), 300 mL per bottle, 0.5% mung bean starch (1.5 g) and 1.34% macadamia nut seed oil (4 g) were added according to the ratio, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, cultured at 28°C for 3 days at 180 rpm, centrifuged to obtain fermentation supernatant and measure viscosity, solid content and pH.

[0056] According to the data, the addition of nutrients such as sucrose will be more helpful to the increase of viscosity, and the increase of oil will also help to increase the viscosity.

[0057] Example 5

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

[0059] (2) Fresh mycelium and water were homogenized at a ratio of 1:5 (mass ratio), 300 mL per bottle, 0.5% sucrose (1.5 g) was added according to the ratio, sterilized at 121°C for 15 min, inoculated with 10 mL of YSC-1 seed liquid, cultured at 28°C for 3 days at 180 rpm, centrifuged to obtain fermentation supernatant and measure viscosity, solid content and pH.

[0060] Example 5.1

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

[0062] (2) Fresh mycelium and water were homogenized at 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 liquid, 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.

[0063] Example 5.2

[0064] (1) The mycelium was prepared as in Example 1.

[0065] (2) Fresh mycelium and water were homogenized at a ratio of 1 :5 (mass ratio), 300 mL per bottle, 0.5% sucrose (1.5 g) was added according to the ratio, 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 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 mushroom seed liquid 1 instead of white mushroom seed liquid 2. The viscosity of the final fermentation liquid 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 prepared in step (1) is stored for 25 days before secondary fermentation. The viscosity of the final fermentation liquid 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 prepared in step (1) is stored for 25 days before secondary fermentation. The viscosity of the final fermentation liquid is 1125 Pa-s.

[0072] Comparative Example 2.1

[0073] Liquid fermentation culture of white mushroom using 5% potato homogenate as substrate (5% potato and water homogenate, 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 fermentation liquid is 224 Pa-s.

[0074] Comparative Example 2.2

[0075] The liquid fermentation culture of white shiitake mushroom was carried out using 1% potato homogenate as substrate (1% potato and water homogenate liquid was divided into three flasks, 300 mL per flask, sterilized at 121℃ for 15 min, then inoculated with 10 mL of YSC-1 seed liquid, cultured at 28℃ for 3 days at 180 rpm), and the viscosity of the obtained fermentation broth was 58.5 Pa·s.

[0076] Comparative Example 3

[0077] The liquid fermentation culture of white shiitake mushroom was carried out using 1% rice as substrate (1% rice was added to the flask, 300 mL of water was added to each flask, sterilized at 121℃ for 15 min, then inoculated with 10 mL of YSC-1 seed liquid, cultured at 28℃ for 3 days at 180 rpm), and the viscosity of the obtained fermentation broth was 170 Pa·s, with an unpleasant odor.

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

[0079] The fermentation supernatant obtained in Examples 1-5 and Comparative Examples 1-3 was 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] As can be seen, the viscosity of the fermentation broth obtained by secondary fermentation with mycelium as substrate is increased, and further supplementing nutrients such as sucrose in the secondary fermentation will further help to increase the viscosity of the fermentation broth, and increasing the oil will also help to increase the viscosity of the fermentation broth; while the fermentation broth obtained by conventional white shiitake mushroom liquid fermentation in Comparative Examples 2.1-3 has lower viscosity. At the same time, the proportion of mycelium in the substrate is different, and the viscosity of the fermentation broth is different.

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

[0084] Table 2 Skin feeling test score table of the fermentation broth obtained in Example 3 and Comparative Example 2.1

[0085]

[0086] As can be seen, the mycelium fermentation broth is smoother than the potato homogenate fermentation broth; the potato homogenate fermentation broth is lighter than the mycelium fermentation broth. Both of them are not tight and not muddy after use.

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

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

[0089] (2) The fermentation supernatant added with mung bean starch has a faint unpleasant odor after sterilization, and the rice white ginseng fermentation broth in Comparative Example 3 also has an unpleasant odor.

[0090] (3) The above-mentioned secondary fermentation process is relatively difficult to implement in mass production; the separation and preservation of mycelium are not easy; and the energy consumption is large.

[0091] Therefore, the inventor continues to explore and obtains another improved embodiment:

[0092] In-situ fermentation process, that is, the mycelium obtaining stage is used as the first step of fermentation, and the obtained mycelium and fermentation broth are not collected separately, but are directly subjected to subsequent treatment together, and the second step of fermentation is directly performed. See the following examples for details.

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

[0094] Example 6 uses the above-mentioned in-situ fermentation process for secondary fermentation, Comparative Examples 4 and 5 do not use the in-situ fermentation process, and Examples 7-8 supplement some components as the second fermentation substrate during the second fermentation process.

[0095] Example 6

[0096] 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 of homogenate and place it in a flask, sterilize at 121℃ for 15min, inoculate with 10mL of YSC-1 seed liquid, cultivate at 28℃, 180rpm for 2 days, then pour out and homogenize the whole, pour the homogenate back into the flask for sterilization and secondary inoculation with 10mL of YSC-1 seed liquid, cultivate at 28℃, 180rpm for 3 days. Centrifuge, take the fermentation supernatant, sterilize. The viscosity of the supernatant is 912.5Pa·s, the solid content is 3.1%, and the pH is 4.98.

[0097] Comparative Example 4

[0098] Take 18 g of fresh potatoes and 18 g of sucrose into 900 mL of water homogenate (potato accounted for 2% and sucrose accounted for 2%), take 300 mL into a flask, sterilize at 121 ℃ for 15 min, inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 2 days, then homogenate the whole, pour the homogenate back into the flask. Add 1.34% of white pool seed oil (4 g / 300 mL), sterilize and inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 3 days. Centrifuge, take the fermentation supernatant, sterilize. The supernatant viscosity is 1363 Pa·s, the solid content is 2.9%, and the pH is 4.97.

[0099] Comparative Example 5

[0100] Take 18 g of fresh potatoes and 18 g of sucrose into 900 mL of water homogenate (potato accounted for 2% and sucrose accounted for 2%), take 300 mL into a flask, sterilize at 121 ℃ for 15 min, inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 2 days, then homogenate the whole, pour the homogenate back into the flask. Add 1.34% of white pool seed oil (4 g / 300 mL), sterilize and inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 3 days. Centrifuge, take the fermentation supernatant, sterilize. The supernatant viscosity is 1363 Pa·s, the solid content is 2.9%, and the pH is 4.97.

[0101] By comparing Example 6, Comparative Example 4 and Comparative Example 5, it can be seen that after the second fermentation, the viscosity of the final fermentation liquid is increased (Comparative Example 5 viscosity 104.5 Pa·s, Comparative Example 4 viscosity 366 Pa·s after secondary fermentation, Example 6 viscosity 912.5 Pa·s after homogenization step and secondary fermentation, the homogenization operation before the second fermentation is essential and is an important step to increase the viscosity of the final fermentation system. The direct sterilization has less damage to the mycelium than the homogenization operation, resulting in a much lower viscosity of the fermentation liquid in Comparative Example 4 than in Example 6.

[0102] Example 7

[0103] Take 18 g of fresh potatoes and 18 g of sucrose into 900 mL of water homogenate (potato accounted for 2% and sucrose accounted for 2%), take 300 mL into a flask, sterilize at 121 ℃ for 15 min, inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 2 days, then homogenate the whole, pour the homogenate back into the flask. Add 1.34% of white pool seed oil (4 g / 300 mL), sterilize and inoculate 10 mL of YSC-1 seed liquid, 28 ℃, 180 rpm, culture for 3 days. Centrifuge, take the fermentation supernatant, sterilize. The supernatant viscosity is 1363 Pa·s, the solid content is 2.9%, and the pH is 4.97.

[0104] Example 8

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

[0106] The photos of the fermentation supernatants prepared in Examples 6-8 are shown in Figure 2 As can be seen, the samples of Examples 6-8 are all yellow to dark yellow turbid fermentation broth, and the samples of Examples 7 and 8 are more turbid and thicker.

[0107] Efficacy study

[0108] 1. DPPH radical scavenging ability test

[0109] Freeze-dry the fermentation supernatants obtained in Example 6 and Comparative Example 2.1 to obtain freeze-dried powders, which are named in-situ fermentation sample and ordinary fermentation sample respectively, and conduct the following efficacy study on them.

[0110] The experimental method refers to the literature (Wu D, Liu P, Li M, Wang C, Zhao D, Zhang JC. Evaluation of the in vitro antioxidant and anti-aging efficacy 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 DPPH· solution with a concentration of 4 mg / mL using anhydrous ethanol, add 2 mL of different concentrations of samples to 2 mL of DPPH· solution, mix well, stand for 30 min, centrifuge at 3000 r / min for 10 min, and measure the absorbance of the supernatant at 517 nm. The ability of the test substance to scavenge DPPH· can be represented by the clearance rate, and the greater the clearance rate, the stronger the scavenging ability. The formula is: clearance rate I = [I - (A i -A j ) / A0] x 100%; where: A i -2 mL of DPPH· solution + 2 mL of sample; A j -2 mL of DPPH· solution + 2 mL of sample; A0-2 mL of DPPH· solution + 2 mL of sample.

[0111] Figure 3 (a) and (b) in FIG. 1 show the DPPH free radical scavenging ability curves of the in-situ fermentation sample and the ordinary fermentation sample respectively. From Figure 3It can be seen that the DPPH free radical scavenging capacity of the in-situ fermentation sample increases with the increase of the concentration, and the free radical scavenging rate is close to 90% at 4000 mg / mL; the DPPH free radical scavenging capacity of the ordinary fermentation sample also increases with the increase of the concentration, but the scavenging rate is below 35%.

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

[0113] 2.1 Mask liquid evaluation

[0114] Two samples are: Example 6 (factory in-situ fermentation material, i.e. fermentation supernatant directly prepared by the process of Example 6, sample label: SMF-factory), Comparative Example 2.1 (5% potato homogenate directly fermented by 290 to prepare fermentation supernatant, sample label: original process).

[0115] (1) Comparison between the two samples, see Figure 4 In terms of product appearance, the sample "SMF-factory" has thicker texture and longer silk; the sample "original process" is more transparent; in use: the sample "SMF-factory" is easier to apply, smoother to apply, and has stronger product oil feeling; after use: the sample "SMF-factory" has brighter skin / skin with stronger oil feeling (immediately), and stronger skin water feeling (2 minutes); overall, the two water products differ greatly in sensory properties: the sample "SMF-factory" has thicker texture, longer silk, is easier to apply, smoother to apply, has stronger product oil feeling, and has brighter skin / skin with stronger oil feeling (immediately) and stronger skin water feeling (2 minutes) after use; the sample "original process" is more transparent.

[0116] (2) Comparison of the above two samples with a certain commercially available fermentation mask (natural original fermentation mask), see Figure 5In terms of product appearance, sample "SMF-Factory" was more transparent, thickest, and had the longest silk threads; sample "Original Process" was the most transparent, thinnest, and had longer silk threads; sample "Natural Original Fermentation Mask" was the least transparent, thickest, and had the shortest silk threads. In use: sample "SMF-Factory" was easier to apply, applied more smoothly, applied thicker, had a stronger skin cooling sensation, and had a stronger product oiliness; sample "Original Process" was somewhat easier to apply, applied somewhat more smoothly, and had the weakest product oiliness; sample "Natural Original Fermentation Mask" was the least easy to apply, applied the least smoothly, and had a somewhat stronger product oiliness. After use: sample "SMF-Factory" had a stronger skin water sensation (2 minutes); sample "Natural Original Fermentation Mask" had a somewhat brighter skin (2 minutes). Overall, the three water-based products differed greatly in sensory attributes: sample "SMF-Factory" (product of Example 6) was more transparent, thickest, and had the longest silk threads, was easier to apply, applied more smoothly, applied thicker, had a stronger skin cooling sensation, had a stronger product oiliness, and had a stronger skin water sensation (2 minutes) after use; sample "Original Process" (product of Comparative Example 2.1) was the most transparent, thinnest, and had longer silk threads, was somewhat easier to apply, applied somewhat more smoothly, and had the weakest product oiliness; sample "Natural Original Fermentation Mask" (commercial product) was the least transparent, thickest, and had the shortest silk threads, was the least easy to apply, applied the least smoothly, had a somewhat stronger product oiliness, and had a somewhat brighter skin (2 minutes) after use.

[0117] 2.2 Emulsion Evaluation

[0118] The two samples were: a fermented supernatant sample prepared in Example 7 (sample label: post-emulsification sample 1), and a fermented supernatant sample prepared in Example 8 (sample label: post-emulsification sample 2).

[0119] (1) Comparison between the two samples, see Figure 6 In terms of product appearance, post-emulsification sample 1 was more transparent; in use, the two water-based products had no difference; after use, post-emulsification sample 2 had a brighter skin (immediately); overall, the two water-based products had little difference in sensory attributes, except that post-emulsification sample 1 was more transparent and post-emulsification sample 2 had a brighter skin (immediately) after use.

[0120] (2) Comparison between the above two samples and a certain commercial emulsion (sample label: essence cream), see Figure 7In terms of product appearance, the serum emulsion was the least transparent. In use, the serum emulsion was the least easy to spread, the least smooth to spread, and the least smooth skin / stronger skin moisture after use (1 minute). Overall, there was no difference between the post-emulsification sample 1 and the post-emulsification sample 2 in sensory attributes, and there was a significant difference between the post-emulsification sample 1, the post-emulsification sample 2, and the serum emulsion in sensory attributes: the post-emulsification sample 1 was smoother skin after use (2 minutes) compared with the serum emulsion; the serum emulsion was the least transparent, the least easy to spread in use, the least smooth to spread, and the least smooth skin / stronger skin moisture after use (1 minute) compared with the post-emulsification sample 1 and the post-emulsification sample 2.

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

[0122] Test principle: whether the sample stimulation is improved compared with the blank control group without sample treatment is compared by determining the COL-I content of human skin fibroblasts. The determination of COL-I is carried out by the method of ELISA kit. The kit is purchased from Cloud-Clone, batch number SEA571Hu, named collagen type I (COL1) detection kit (enzyme-linked immunosorbent assay).

[0123] The experiment includes a blank control group (Control, no sample and cells), a sample group (Samples, with sample and cells), and a positive control group (Positive control, TGF-β as a positive control, 100 ng / mL). Well-grown human skin fibroblasts (HSFs) are selected, and the cell number is controlled at 1.5×10 6 mL -1 per hole, and then cultured in a 37°C, 5% CO2 incubator overnight, treated with samples for 72 h, collected cell supernatant, and detected the COL-I (collagen I, the higher the value) content of the supernatant. The index is 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., the fermentation supernatant directly prepared by the process of Example 6, sample label: SMF), Comparative Example 2.1 (fermentation supernatant obtained by directly 290 fermenting 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 are 5% sample concentration (i.e., a solution sample prepared by diluting the fermentation liquid to a concentration of 5%), and TGF-β is used as a positive control at 100 ng / mL.

[0125] The results are shown in Table 1. Figure 8It can be seen that: compared with the Control group, TGF-β significantly promotes the production of COL-I; the sample "original process" and the sample "SMF" also significantly increase the production of COL-I, and there is no significant difference compared with the positive control TGF-β; the post-emulsification sample 1 and the post-emulsification sample 2 also have the effect of significantly increasing the production of COL-I, but significantly decrease compared with the positive control. Therefore, the sample "SMF" and the sample "original process" have better effects in promoting the production of COL-I.

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

[0127] AQP3 is water channel protein 3, which is related to the transport of water molecules, and an increase in the value indicates that it helps water enter the cell and is related to moisturizing effect. The AQP3 assay uses the method of an ELISA kit. The kit is purchased from Cloud-Clone, batch number SEA581Hu, and the name is: Water Channel Protein 3 (AQP3) Detection Kit (ELISA method).

[0128] The experimental setup includes a blank control group (Control, no sample with cells), a sample group (Samples, with sample with cells), and a positive control group (Positive control, Ciglitazone, Cig, 5 μM). Choose well-grown HaCaT cells, control the cell number to be 1.5 × 10 6 mL -1 , spread in a 6-well plate, 2 mL per well, then incubate in a 37℃, 5% CO2 incubator overnight, add samples for 48h, collect cells, and lyse adherent cells with lysis buffer, centrifuge and take the supernatant to be stored in a -20℃ refrigerator for detection of intracellular AQP3 content. The index is the ratio of the AQP3 content of each treatment group to the AQP3 content of the Control group.

[0129] The samples of this experiment are: Example 6 (factory-produced in-situ fermentation material body, i.e. fermentation supernatant, sample label: SMF), Comparative Example 2.1 (fermentation supernatant obtained by directly 290 fermenting 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 are 5% sample concentration, and Ciglitazone (Cig, 5 μM) is used as a positive control.

[0130] The results are shown in Figure 9It can be seen that, compared with the Control group, Cig significantly promotes the production of AQP3; the sample "original process" and the sample "SMF" also significantly increase the production of AQP3, and the sample "SMF" has no significant difference compared with the positive control Cig; the post-emulsification sample 1 and the post-emulsification sample 2 also have a significant effect on increasing the production of AQP3, and have no significant difference compared with the positive control. Therefore, in terms of promoting the production of AQP3, the sample "SMF", the post-emulsification sample 1 and the post-emulsification sample 2 have better effects.

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

[0132] Zebrafish skin has a similar inflammatory response to human skin. After the skin is subjected to inflammatory stimulation, it can induce the expression of a large number of related inflammatory factor genes. A zebrafish inflammation induction model is constructed using copper sulfate. After the test substance is administered, the relative expression of inflammatory-related factor genes (IL-1β, IL-6, NF-κB and TNFα) is detected to evaluate whether the cosmetic has soothing and anti-inflammatory effects.

[0133] Zebrafish embryos developed to day 3 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 main instruments and equipment include: a real-time PCR instrument (Applied Biosystems QuantStudio 5), a low-temperature refrigerated centrifuge, a micro spectrophotometer (Thermo Scientific Nanodrop 2000), an analytical balance (0.01 g), pipettes, a refrigerator, a clean bench, a zebrafish culture system, and a constant temperature incubator.

[0135] The specific experimental procedure is as follows: A blank control group, a model group (1 μM), a positive control group (0.02 g / L), and a sample group (5.0 g / L, prepared by adding water to fermentation broth) were set up. Each experimental group had three replicates, with 10 zebrafish embryos per replicate, placed in 24-well plates. 2 mL of the working solution corresponding to each concentration group was added to each well. The 24-well plates were placed in a constant temperature incubator at 28.5 ± 0.5℃ for 24 hours before sampling. Total RNA was extracted from the zebrafish in each group, reverse transcribed, and amplified by quantitative real-time PCR. β-actin gene was used as an internal reference gene. -△△Ct The relative expression levels of inflammatory cytokine 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 was considered statistically significant.

[0137] The test results are as follows: Figure 10 The relative expression levels of zebrafish inflammatory cytokine genes in each group are shown. It can be seen that, compared with the model group, the expression of zebrafish inflammatory cytokine genes (IL-1β, IL-6, NF-κB, and TNFα) was inhibited by the samples at a concentration of 5.0 g / L by 53.97%, 39.54%, 52.12%, and 37.86%, respectively, which were statistically significant (p<0.05) (see [link to relevant documentation]). Figure 10 (See the figure above). The positive control group showed inhibition rates of 26.99%, 35.06%, 20.91%, and 30.35% on the expression of zebrafish inflammatory factor genes, respectively. That is, under the experimental conditions, sample "SMF-2" (the second batch of material reproduced according to the method in Example 6) at a concentration of 5.0 g / L could significantly inhibit the expression of zebrafish inflammatory factor genes (p<0.05), and had a soothing effect.

[0138] As a comparison, the inventors also studied the soothing effect of the fermentation broth sample of Paecilomyces cicadae (i.e. Cordyceps cicadae CH2347, accession number CGMCC No. 40399, deposited on April 10, 2023, classified as Cordyceps cicadae CH2347, deposited at China General Microbiological Culture Collection Center, abbreviated as CGMCC, located at No. 1, Michenxi Li, Beijing, China, with a postal code of 100101). The details are as follows: the medium formula is 2% potato + 2% sucrose. First, a mixed solution containing 2% sucrose was prepared at 300 mL, then 6 g of fresh peeled potato was added to a homogenizer, and after adding the above prepared 300 mL solution, homogenization was carried out, then it was all transferred into a 500 mL capacity triangular flask, sterilized, inoculated with 20 mL of Paecilomyces cicadae seed liquid, and fermented at 28°C for 3 days at 180 rpm. After homogenization, it was all sterilized again (121°C for 20 min) and inoculated with 20 mL of Paecilomyces cicadae seed liquid, and fermented at 28°C for 3 days at 180 rpm. The supernatant (fermentation broth) was obtained by centrifugation, sterilized (121°C for 20 min), and after adding 1% preservative (the preservative system is xianxian ketone and hexanediol 1:1), sample No. 300 was obtained. The sample No. 300 was tested for zebrafish inflammatory factor gene expression inhibition 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 a significant difference (p<0.05) Figure 10 The following figure), and 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" at a concentration of 5.0 g / L can significantly inhibit the expression of zebrafish inflammatory factor genes (IL-1β, IL-6, NF-κB and TNFα) (p<0.05), and also has a soothing effect, but its inhibition rate is significantly lower than that of the sample of the present application.

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

[0140] The zebrafish skin has a similar inflammatory stimulus response to human skin. After the skin is subjected to inflammatory stimulation, neutrophils can be induced to aggregate on the surface of the skin. A copper sulfate-induced zebrafish inflammation model was constructed, and 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 developed to the third day 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 main instruments and equipment included a body microscope, a zebrafish breeding system, an analytical balance (one ten-thousandth), a pipette, a refrigerator, a clean bench, a constant temperature incubator, etc.

[0142] The specific test procedure was 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, 3 repeats were set up for each experimental group, 10 zebrafish embryos were set up for each repeat, and they were placed in a 24-well plate, 2 mL of 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 4 h of incubation. After the incubation was completed, not less than 12 zebrafish embryos were randomly selected from each group, and they were fixed with 4% paraformaldehyde overnight. The larvae were washed with PBS 4 times, 10 min each time. Sudan black solution was added for staining, and incubation was carried out in the dark for 40 min. The fish were washed with 70% ethanol under dark conditions until the body color was transparent, and then washed with PBS 3 times. The fish were uniformly adjusted to the lateral body position, and photographed using a body microscope. The number of neutrophils on the surface of the skin in the specific area of each fish was counted.

[0143] The neutrophil aggregation inhibition rate was calculated, and the formula was as follows: inhibition rate (%) = (M-T) / M x 100%; in the formula: T was the average number of neutrophils in the specific area of the fish embryos in the test group; M was the average number of neutrophils in the specific area of the fish embryos in the model group; a two-tailed T test was performed on the neutrophils in the specific area of the fish embryos in the test group and the neutrophils in the specific area of the fish embryos in the model group, and p<0.05 was considered to have significant difference.

[0144] The detection results were as follows: the mean analysis of the neutrophils in the specific area of the zebrafish embryos in each group was shown in the upper graph of Figure 11 The lower graph of Figure 11 was a typical graph of soothing anti-inflammatory (the red dashed line part was the quantitative counting area, and the red arrow was the neutrophil). It could be seen that compared with the model group, the sample at a concentration of 5.0 g / L had an inhibition rate of 88.85% on the aggregation of neutrophils in the specific area of the zebrafish, and there was a significant difference (p<0.05). Under the experimental conditions in this experiment, the sample “SMF-2” (the second batch of material prepared according to the method of Example 6) could significantly inhibit the aggregation of neutrophils in the specific area of the zebrafish (p<0.05), and had a soothing effect.

[0145] Similarly, the neutrophil inhibition rate test of the above sample 300 was carried out according to the above method, and the results were shown in Figure 12The upper and lower graphs of Figure 1 show that, compared with the model group, the sample 300 had an inhibition rate of 75.09% on the aggregation of neutrophils in the specific area of zebrafish at a concentration of 5.0 g / L, and had a significant difference (p<0.05). Under the experimental conditions of this experiment, the sample 300 can significantly inhibit the aggregation of neutrophils in the specific area of zebrafish (p<0.05), and has a soothing effect, but its inhibition rate is also significantly lower than that of the sample of the present 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, fresh 290 mycelium was used as the main substrate for fermentation, and the preparation method of the 290 mycelium was the same as that of Example 1.

[0148] Example 9

[0149] The fresh 290 mycelium was weighed, water was added at a ratio of 1:10, and the slurry was divided into triangular bottles, each bottle containing 300 mL. After sterilization at 121°C for 30 min, 290 seed liquid was inoculated at an inoculation amount of 10 mL, and fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0150] Example 10

[0151] The fresh 290 mycelium was weighed, water was added at a ratio of 1:10, and the slurry was divided into triangular bottles, each bottle containing 300 mL. After adding sucrose at a ratio of 0.5%, sterilization was carried out at 121°C for 30 min, 290 seed liquid was inoculated at an inoculation amount of 10 mL, and fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0152] Example 11

[0153] The fresh 290 mycelium was weighed, water was added at a ratio of 1:10, and the slurry was divided into triangular bottles, each bottle containing 300 mL. After adding sucrose at a ratio of 0.5%, sterilization was carried out at 121°C for 30 min, 290 seed liquid was inoculated at an inoculation amount of 10 mL, and fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation. The process of this example is the same as that of Example 10, except that the experimental batch is different.

[0154] Example 12

[0155] Freshly prepared 290 mycelium was weighed, then water was added at a ratio of 1:10 for homogenization, and was divided into flasks, 300 mL per flask. Green bean starch was added at a ratio of 0.5%, and was sterilized at 121°C for 30 min. 290 seed liquid was inoculated, with an inoculation amount of 10 mL. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0156] Example 13

[0157] Freshly prepared 290 mycelium was weighed, then water was added at a ratio of 1:10 for homogenization, and was divided into flasks, 300 mL per flask. Green bean starch was added at a ratio of 0.5%, and was sterilized at 121°C for 30 min. 290 seed liquid was inoculated, with an inoculation amount of 10 mL. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation. The process of this example is the same as that of Example 12, except that the experimental batch is different.

[0158] Example 14

[0159] Freshly prepared 290 mycelium was weighed, then water was added at a ratio of 1:10 for homogenization, and was divided into flasks, 300 mL per flask. 4 g of macadamia nut oil was added, and was sterilized at 121°C for 30 min. 290 seed liquid was inoculated, with an inoculation amount of 10 mL. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0160] Comparative Example 6

[0161] Fresh potatoes were taken at a ratio of 1% by mass, were homogenized with water, and were sterilized at 121°C for 30 min, 300 g per flask. 290 seed liquid was inoculated, with an inoculation amount of 10 mL per flask. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0162] Comparative Example 7

[0163] Fresh potatoes were taken at a ratio of 5% by mass, were homogenized with water, and were sterilized at 121°C for 30 min, 300 g per flask. 290 seed liquid was inoculated, with an inoculation amount of 10 mL per flask. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was obtained by centrifugation.

[0164] Comparative Example 8

[0165] 300 g of material was taken per flask, and rice powder was added at a ratio of 1% by mass, and was sterilized at 121°C for 30 min. 290 seed liquid was inoculated, with an inoculation amount of 10 mL per flask. Fermentation was carried out at 28°C and 180 rpm for 3 days. The supernatant was 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, and the photos are shown in Figure 13 .

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

[0168]

[0169] As can be seen from Table 3, the total sugar content of the samples with high viscosity is not high, and the polypeptide content of the samples tested is low; as can be seen from Figure 13 the photos, the samples of the comparative examples and the examples are all yellowish to yellow, transparent or turbid fermentation broth; the color of Comparative Examples 6-8 is lighter and more transparent; the color of Examples 9-14 is yellowish, more turbid and thicker.

[0170] Comparative Example 9

[0171] First, potato glucose water medium was used, and after sterilization, the fungus Inonotus obliquus seed liquid (laboratory number 415, prepared in the same way as the seed liquid of the white mushroom fungus) was inoculated, the inoculation amount (volume ratio of seed liquid to medium) was 1:30, 28°C, 180 rpm, and after 3 days of culture, the mycelium was collected by centrifugation. Then, the fresh 415 mycelium was weighed, water was added at a ratio of 1:10, and the slurry was divided into triangular bottles, each bottle containing 300 mL, sterilized at 121°C for 30 min, inoculated with 415 seed liquid, the inoculation amount was 10 mL / bottle, 28°C, 180 rpm for 3 days of fermentation, and the supernatant was collected by centrifugation.

[0172] Comparative Example 10

[0173] First, potato glucose water medium was used, and after sterilization, the fungus Inonotus obliquus seed liquid (laboratory number 415, prepared in the same way as the seed liquid of the white mushroom fungus) was inoculated, the inoculation amount (volume ratio of seed liquid to medium) was 1:30, 28°C, 180 rpm, and after 3 days of culture, the mycelium was collected by centrifugation. Then, the fresh 415 mycelium was weighed, water was added at a ratio of 1:10, and the slurry was divided into triangular bottles, each bottle containing 300 mL, sterilized at 121°C for 30 min, inoculated with 415 seed liquid, the inoculation amount was 10 mL / bottle, 28°C, 180 rpm for 3 days of fermentation, and the supernatant was collected by centrifugation.

[0174] Comparative Example 11

[0175] First, potato dextrose aqueous medium was sterilized and inoculated with Inonotus obliquus seed culture (laboratory number 415, seed culture preparation method is the same as that of Inonotus cylindrica seed culture) at a ratio of 1:30. After incubation at 28℃ and 180 rpm for 3 days, the mycelium was collected by centrifugation. Then, freshly prepared 415 mycelium was weighed, and water was added at a ratio of 1:10 to homogenize the mixture. The homogenate was then dispensed into Erlenmeyer flasks, 300 mL per flask. Mung bean starch was added at a ratio of 0.5%, and the mixture was sterilized at 121℃ for 30 min. The 415 seed culture was then inoculated at a ratio of 10 mL, and fermented at 28℃ and 180 rpm for 3 days. The supernatant was collected by centrifugation.

[0176] The viscosity, total sugar, and polypeptide of the samples (containing preservatives) obtained from Comparative Examples 9-11 were tested, and 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 shown in Table 4, the viscosity of the fermentation broth of Inonotus obliquus 415 mycelium is much lower than that of the fermentation broth of Ginseng mycelium in this application, and the total sugar content is lower than that of the fermentation broth of Ginseng mycelium in this application, while the polypeptide content is higher than that of the fermentation broth of Ginseng mycelium in this application.

[0180] Finally, it should be noted that, if any, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0181] Although this disclosure has been described above through specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this disclosure within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this disclosure.

Claims

1. A method for direct production of a high viscosity fermentation emulsion by biofermentation, characterized in that, The method comprises the following steps: Step 1, preparing white ginseng fungus mycelium by liquid fermentation culture; wherein, the preparation method of the white ginseng fungus mycelium comprises: adding 2% of potato and 2% of sucrose into water and then performing homogenization treatment, inoculating white ginseng fungus seed liquid for fermentation culture, and finally separating to obtain the white ginseng fungus mycelium; or the preparation method of the white ginseng fungus mycelium comprises: inoculating white ginseng fungus seed liquid into potato glucose water culture medium for fermentation culture, and finally separating to obtain the white ginseng fungus mycelium; Step 2, configuring the white ginseng fungus mycelium and water into a substrate according to a proportion, sterilizing, inoculating white ginseng fungus seed liquid, performing fermentation culture, centrifuging to obtain supernatant, sterilizing to obtain fermentation liquid; The white ginseng fungus is Schizophyllum commune (Fr.) Schum. Schizophyllum commune ) YSC1, with the preservation number of CGMCC No. 17788; The preparation method of the white ginseng fungus seed liquid comprises the following steps: (1) taking 2-3 solid strains with a diameter of 0.5 cm from the solid strain of white ginseng fungus growing in a PDA plate together with the culture medium, inoculating into 300 mL potato glucose water culture medium, and culturing at 28 DEG C for 3 days at 180 rpm to obtain white ginseng fungus seed liquid 1; (2) homogenizing the white ginseng fungus seed liquid 1 and taking 10 mL as seed liquid to inoculate into 300 mL potato glucose water culture medium, and culturing at 28 DEG C for 3 days at 180 rpm to obtain white ginseng fungus seed liquid 2.

2. The method of claim 1, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation, and The volume ratio of the white ginseng fungus mycelium in the white ginseng fungus seed liquid to the whole liquid culture medium is 75-90%, and the volume ratio of the seed liquid to the substrate is (0.1-5):

30.

3. The method of claim 2, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation, and The volume ratio of the seed liquid to the substrate is 1:

30.

4. The method of claim 2, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation. The volume ratio of the white ginseng fungus mycelium in the white ginseng fungus seed liquid to the whole liquid culture medium is 85-90%.

5. The method of directly producing a high viscosity fermentation emulsion by biofermentation according to any one of claims 1 to 4, characterized in that, In step 2, the mass ratio of the white ginseng fungus mycelium to water in the substrate is 1:5-1:

15.

6. The method of claim 5, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation. In step 2, the mass ratio of the white ginseng fungus mycelium to water in the substrate is 1:5-1:

10.

7. The method of direct production of high viscosity fermentation emulsions by biofermentation according to any one of claims 1 to 4, 6, characterized in that, In step 2, the substrate further comprises sucrose, and the addition amount of sucrose is 0-2%; in step 2, the addition amount of sucrose in the substrate is 0.5%.

8. The method of direct production of high viscosity fermentation emulsions by biological fermentation according to any one of claims 1-4, 6, characterized in that, In step 2, the substrate further comprises mung bean starch, and the addition amount of mung bean starch is 0-3%.

9. The method of claim 8, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation. In step 2, the addition amount of mung bean starch in the substrate is 0.5%.

10. The method of claim 8, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation. In step 2, the substrate further comprises macadamia nut seed oil, and the addition amount of macadamia nut seed oil is 0-3%.

11. The method of claim 10, wherein the high viscosity fermentation emulsion is prepared directly from the biofermentation. In step 2, the addition amount of macadamia nut seed oil in the substrate is 1-1.5%.

12. The method of direct production of high viscosity fermented emulsions by biological fermentation according to any one of claims 1-4, 6, 9-11, characterized in that, In step 1, the liquid fermentation is directly homogenized after the end of the liquid fermentation, and then the homogenized product is used as a substrate for step 2.

13. The method for directly preparing high-viscosity fermented emulsions by biological fermentation according to claim 12, characterized in that, In step 2, the substrate further comprises oil, and the oil is at least one selected from the group consisting of white pool flower seed oil, soybean oil, flaxseed oil and corn germ oil, and the addition amount of the oil is 0.5-3%.

14. The method for directly preparing high-viscosity fermented emulsions by biological fermentation according to claim 13, characterized in that, In step 2, the addition amount of the oil is 1%.

15. The method of directly producing a high viscosity fermentation emulsion by biofermentation according to any one of claims 1-4, 6, 9-11, 13-14, characterized in that, The fermentation culture is performed at a temperature of 24-30 DEG C for 48-96 h.

16. The method of direct production of high viscosity fermented emulsions by biological fermentation according to any one of claims 1-4, 6, 9, 11, 13-14, characterized by, The fermentation culture is performed in a shaking bed, and the rotation speed in the shaking bed is 150 rpm-200 rpm.

17. A high viscosity fermented emulsion product, characterized in that, The method is prepared by the method in any one of claims 1-16.

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