Method for preparing inositol hexakisphosphate of sea rice origin by bidirectional fermentation, fermentation product, cosmetic

Abstract

This application relates to the field of cosmetics, specifically a method for preparing inositol hexaphosphate from sea rice through bidirectional fermentation, the fermentation product, and cosmetics. Sea rice is pulverized to obtain sea rice powder; Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation to obtain a fermentation mixture; the fermentation mixture is centrifuged to remove bacterial cells, yielding the fermentation product; the fermentation product contains Bacillus coagulans lactic acid and inositol hexaphosphate; the release rate of inositol hexaphosphate in the fermentation product is greater than or equal to 33.5%; the lactic acid content in the fermentation product is greater than 0.69 g / L. The high content of these two active ingredients can effectively enhance the growth and migration rate of skin cells, inhibit inflammatory factors, and suppress the growth of skin pathogens. Compared to existing physical extraction methods of active ingredients from sea rice, the bidirectional fermentation method of this application is green and natural, does not produce additional solvent residues, and has low energy consumption and high efficiency.

Description

Technical Field

[0001] This application relates to the field of cosmetics, and more specifically, to a method for preparing inositol hexaphosphate from sea rice through bidirectional fermentation, the fermentation product, and cosmetics. Background Technology

[0002] Sea rice refers to a special type of rice that is not afraid of short-term seawater immersion and can grow in high-salt soils such as saline-alkali land and tidal flats.

[0003] Because sea rice has a high protein and dietary fiber content, it has a poor taste and is not easily digested and absorbed, which limits people's direct consumption of sea rice (brown rice).

[0004] Therefore, other methods are needed to improve the usability of sea rice. The current method for extracting active ingredients from sea rice is physical extraction. However, this physical extraction method has problems such as high energy consumption, low extraction rate, and the need for additional solvents to aid dissolution. Summary of the Invention

[0005] The purpose of this application is to provide a method for preparing inositol hexaphosphate from sea rice through bidirectional fermentation, as well as fermentation products and cosmetics.

[0006] In a first aspect, this application provides a method for bidirectional fermentation of inositol hexaphosphate derived from sea rice, comprising:

[0007] The sea rice grains are crushed to obtain sea rice powder;

[0008] Bacillus coagulans seed culture was inoculated into a fermentation medium and fermented to obtain a fermentation mixture;

[0009] The fermentation mixture was centrifuged to remove the bacterial cells and obtain the fermentation product; the fermentation product contained Bacillus coagulans lactic acid and inositol hexaphosphate.

[0010] The release rate of inositol hexaphosphate in the fermentation products is greater than or equal to 33.5%; the content of lactic acid in the fermentation products is greater than 0.69 g / L.

[0011] The above-mentioned technical solution involves inoculating Bacillus coagulans seed culture into a fermentation medium supplemented with sea rice powder for fermentation. The resulting fermentation product contains Bacillus coagulans lactic acid and inositol hexaphosphate. The release rate of inositol hexaphosphate in the fermentation product is greater than or equal to 33.5%, and the lactic acid content is greater than 0.69 g / L. This method allows the fermentation product to be rich in both lactic acid and inositol hexaphosphate. The simultaneous enrichment of these two active ingredients can effectively enhance the growth and migration rate of skin cells, inhibit inflammatory factors, and suppress the growth of skin pathogens.

[0012] Furthermore, compared to the existing physical extraction methods used for extracting active ingredients from sea rice, the two-way fermentation method of this application is green and natural, does not produce additional solvent residues, and has low energy consumption and high efficiency.

[0013] In other embodiments of this application, the Bacillus coagulans seed culture inoculated into the culture medium accounts for 2% to 8% of the fermentation culture medium volume by volume percentage.

[0014] In other embodiments of this application, the amount of sea rice powder added to the fermentation culture medium is 2% to 8% by mass percentage.

[0015] In other embodiments of this application, Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation, including:

[0016] The Bacillus coagulans seed culture was inoculated into the fermentation medium and fermented for 48-72 hours.

[0017] In other embodiments of this application, the fermentation medium consists of: 2%–8% sea rice powder, 0.1%–1.0% magnesium sulfate, with the remainder being water and Bacillus coagulans seed culture.

[0018] In other embodiments of this application, the pH of the fermentation medium is 7.5 to 8.5.

[0019] In other embodiments of this application, the preparation of Bacillus coagulans seed solution includes:

[0020] Transfer single colonies of Bacillus coagulans to liquid culture medium and culture for 24-48 hours.

[0021] In other embodiments of this application, the liquid culture medium comprises, by weight percentage: 1.0%–2.0% tryptone, 0.5%–1.5% yeast extract, 0.1%–1.0% magnesium sulfate, and the balance being water;

[0022] In other embodiments of this application, the pH of the liquid culture medium is 7.5 to 8.5.

[0023] In other embodiments of this application, Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation, including:

[0024] The Bacillus coagulans seed culture was inoculated into the fermentation medium and fermented at 35℃~38℃ and 230rpm~250rpm.

[0025] Secondly, this application provides a fermentation product prepared by the bidirectional fermentation method for preparing rice-derived inositol hexaphosphate provided in any of the first aspects above.

[0026] Thirdly, this application provides a cosmetic product comprising the six fermentation products provided in the second aspect above. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0028] Therefore, the following detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0029] This application provides a method for bidirectional fermentation of inositol hexaphosphate derived from sea rice, comprising:

[0030] The sea rice grains are crushed to obtain sea rice powder;

[0031] Bacillus coagulans seed culture was inoculated into a fermentation medium and fermented to obtain a fermentation mixture;

[0032] The fermentation mixture was centrifuged to remove the bacterial cells and obtain the fermentation product; the fermentation product contained Bacillus coagulans lactic acid and inositol hexaphosphate.

[0033] By mass percentage, the release rate of inositol hexaphosphate in the fermentation product is greater than or equal to 33.5%; the content of lactic acid in the fermentation product is greater than 0.69 g / L.

[0034] The above-mentioned technical solution involves inoculating Bacillus coagulans seed culture into a fermentation medium supplemented with sea rice powder for fermentation. The resulting fermentation product contains Bacillus coagulans lactic acid and inositol hexaphosphate. The release rate of inositol hexaphosphate in the fermentation product is greater than or equal to 33.5%, and the lactic acid content is greater than 0.69 g / L. This method allows the fermentation product to be rich in both lactic acid and inositol hexaphosphate. The simultaneous enrichment of these two active ingredients can effectively enhance the growth and migration rate of skin cells, inhibit inflammatory factors, and suppress the growth of skin pathogens.

[0035] Furthermore, in some embodiments of this application, the Bacillus coagulans seed culture inoculated into the culture medium accounts for 2% to 8% of the volume of the fermentation culture medium, by volume percentage.

[0036] For example, by volume percentage, the Bacillus coagulans seed culture inoculated into the culture medium accounts for 2%, 2.2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 3%, 3.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8% of the fermentation culture medium volume, or any range between two of the aforementioned values.

[0037] A large number of Bacillus coagulans can rapidly decompose and consume sea rice within a certain timeframe, releasing a significant amount of inositol hexaphosphate (IHP). However, exceeding the fermentation system's capacity leads to an overabundance of Bacillus coagulans. As nutrients are depleted, a considerable portion of the IHP is decomposed and utilized by Bacillus coagulans, ultimately resulting in a decrease in IHP content. Experimental studies have shown that an inoculum size of 2%-8% is more conducive to increasing the IHP content in the fermentation broth while simultaneously promoting lactic acid production by Bacillus coagulans.

[0038] Furthermore, in some embodiments of this application, the amount of sea rice powder added to the fermentation culture medium is 2% to 8% by mass percentage.

[0039] For example, in some embodiments of this application, the amount of sea rice powder added to the fermentation culture medium, by mass percentage, is 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, or any two of the aforementioned values.

[0040] The amount of sea rice added to the culture medium significantly affects the growth of Bacillus coagulans using nutrients from sea rice and the promotion of inositol hexaphosphate release. Too high a proportion of sea rice may exceed the decomposition capacity of Bacillus coagulans, failing to increase the inositol hexaphosphate content. Too low a proportion may not be sufficient to promote greater inositol hexaphosphate release. Empirical studies have shown that a sea rice content of 2%-8% in the culture medium is more conducive to increasing the inositol hexaphosphate content in the fermentation broth, while simultaneously promoting lactic acid production by Bacillus coagulans.

[0041] Furthermore, in some embodiments of this application, Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation, including:

[0042] The Bacillus coagulans seed culture was inoculated into the fermentation medium and fermented for 48-72 hours.

[0043] Exemplary, in some embodiments of this application, Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation, including:

[0044] Inoculate the Bacillus coagulans seed culture into the fermentation medium and ferment for 48h, 50h, 52h, 55h, 58h, 60h, 62h, 65h, 66h, 68h, 70h, 72h or any two of the aforementioned values.

[0045] Fermentation time affects the growth of Bacillus coagulans using nutrients in sea rice and promotes the release of inositol hexaphosphate. When the fermentation time is too short or too long, the levels of lactic acid and inositol hexaphosphate in the fermentation products decrease. Experimental studies have shown that a fermentation time of 48-72 hours is more conducive to increasing the inositol hexaphosphate content in the fermentation broth and simultaneously promoting lactic acid production by Bacillus coagulans.

[0046] Furthermore, in some embodiments of this application, the fermentation culture medium consists of: 2%–8% sea rice powder, 0.1%–1.0% magnesium sulfate, with the remainder being water and Bacillus coagulans seed culture.

[0047] For example, in some embodiments of this application, the fermentation culture medium consists of: 2%, 3%, 4%, 5%, 6%, 7%, 8% or any two of the aforementioned values ​​of sea rice powder, 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or any two of the aforementioned values ​​of magnesium sulfate; the balance being water and Bacillus coagulans seed culture.

[0048] Furthermore, in some embodiments of this application, the pH of the fermentation medium is 7.5 to 8.5.

[0049] For example, in some embodiments of this application, the pH of the fermentation medium is 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or a range between any two of the aforementioned values.

[0050] Furthermore, in some embodiments of this application, the preparation of Bacillus coagulans seed solution includes:

[0051] Transfer single colonies of Bacillus coagulans to liquid culture medium and culture for 24-48 hours.

[0052] Exemplary, in some embodiments of this application, the preparation of Bacillus coagulans seed solution includes:

[0053] Single colonies of Bacillus coagulans were transferred to liquid culture medium and cultured for 24h, 26h, 28h, 30h, 32h, 36h, 38h, 40h, 42h, 46h, 48h, or any two of the aforementioned values.

[0054] Furthermore, in some embodiments of this application, the liquid culture medium comprises, by mass percentage: 1.0%–2.0% tryptone, 0.5%–1.5% yeast extract, 0.1%–1.0% magnesium sulfate, and the balance being water.

[0055] For example, the liquid culture medium, by weight percentage, comprises: 1.0%, 1.2%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0% or any two of the aforementioned values ​​of tryptone; 0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.5% or any two of the aforementioned values ​​of yeast extract; 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% or any two of the aforementioned values ​​of magnesium sulfate; and the balance being water.

[0056] Furthermore, in some embodiments of this application, the pH of the liquid culture medium is 7.5 to 8.5.

[0057] For example, in some embodiments of this application, the pH of the liquid culture medium is 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 or a range between any two of the aforementioned values.

[0058] Furthermore, in some embodiments of this application, Bacillus coagulans seed culture is inoculated into a fermentation medium for fermentation, including:

[0059] The Bacillus coagulans seed culture was inoculated into the fermentation medium and fermented at 35℃~38℃ and 230rpm~250rpm.

[0060] For example, in some embodiments of this application, Bacillus coagulans seed liquid is inoculated into a fermentation medium and fermented at 35°C, 36°C, 37°C, 38°C or any two of the aforementioned values, and at 230 rpm, 235 rpm, 240 rpm, 245 rpm, 248 rpm, 250 rpm or any two of the aforementioned values.

[0061] Some embodiments of this application provide a fermentation product prepared by a method for preparing rice-derived inositol hexaphosphate using bidirectional fermentation as described in any of the foregoing embodiments.

[0062] Some embodiments of this application provide a cosmetic product including the fermentation product provided in any of the foregoing embodiments.

[0063] In some embodiments of this application, the cosmetics described above also include cosmetic carriers acceptable in the art.

[0064] The aforementioned cosmetics, by adding the aforementioned fermentation products, can effectively enhance the growth and migration rate of skin cells, inhibit inflammatory factors, and suppress the growth of skin pathogens.

[0065] The features and performance of this application will be further described in detail below with reference to embodiments:

[0066] Example 1

[0067] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0068] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0069] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0070] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 91.5% distilled water, pH 8.0;

[0071] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0072] Example 2

[0073] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0074] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0075] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0076] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 93.5% distilled water, and the balance Bacillus coagulans fermentation seed liquid obtained in step (2); pH 8.0;

[0077] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 2% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium, and fermentation was carried out at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0078] Example 3

[0079] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0080] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0081] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0082] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 87.5% distilled water, pH 8.0;

[0083] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 8% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0084] Example 4

[0085] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0086] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0087] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0088] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 2% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 93.5% distilled water, pH 8.0;

[0089] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0090] Example 5

[0091] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0092] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0093] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0094] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 8% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 87.5% distilled water, pH 8.0;

[0095] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0096] Example 6

[0097] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0098] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0099] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0100] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 91.5% distilled water, pH 8.0;

[0101] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium, and fermentation was carried out at 37°C and 240 rpm. After fermentation for 48 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0102] Example 7

[0103] A method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation is provided, comprising the following steps:

[0104] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through an 80-mesh sieve to obtain sea rice powder for later use;

[0105] (2) Activation of Bacillus coagulans strain: Single colonies of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0106] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 91.5% distilled water, pH 8.0;

[0107] (4) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium, and fermentation was carried out at 37°C and 240 rpm. After fermentation for 72 h, the fermentation mixture was centrifuged to remove the bacterial cells, and a fermentation product rich in lactic acid and inositol hexaphosphate derived from sea rice was obtained.

[0108] Comparative Example 4

[0109] A fermentation product of Bacillus coagulans is prepared according to the following steps:

[0110] (1) A single colony of Bacillus coagulans (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.645) was transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.5% tryptone, 1% yeast extract, 0.5% magnesium sulfate, and 97% distilled water, adjusted to pH 8.0) and cultured for 24 hours to obtain Bacillus coagulans fermentation seed liquid;

[0111] (2) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 1% yeast extract, 0.5% magnesium sulfate, 94.5% distilled water, pH 8.0;

[0112] (3) Fermentation of sea rice by Bacillus coagulans: Based on the total volume of the fermentation medium obtained in step (2) above, 4% of the Bacillus coagulans fermentation seed liquid obtained in step (1) was inoculated into the fermentation medium obtained in step (2) above, and fermentation was carried out at 37°C and 240 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells and obtain the fermentation product.

[0113] Comparative Example 1

[0114] A brewing yeast fermentation product is provided, prepared according to the following steps:

[0115] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through a 200-mesh sieve to obtain sea rice powder for later use;

[0116] (2) Activation of Saccharomyces cerevisiae strain: A single colony of Saccharomyces cerevisiae (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 2.90) was transferred to a liquid culture medium (by mass percentage, the liquid culture medium consisted of 0.5% peptone, 1.0% glucose, 0.3% yeast extract, and 98.2% distilled water) and cultured for 24 hours to obtain Saccharomyces cerevisiae fermentation seed liquid;

[0117] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% magnesium sulfate, 91.5% distilled water, and pH natural (about 6.0);

[0118] (4) Fermentation of sea rice by Saccharomyces cerevisiae: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Saccharomyces cerevisiae fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 28°C and 220 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the cells and obtain the fermentation product.

[0119] Comparative Example 2

[0120] A fermentation product of Bacillus subtilis subtilis is provided, prepared according to the following steps:

[0121] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through a 200-mesh sieve to obtain sea rice powder for later use;

[0122] (2) Activation of Bacillus subtilis subsp. subtilis strain: Single colonies of Bacillus subtilis subsp. subtilis (purchased from Guangdong Provincial Microbial Culture Collection Center, number GDMCC 1.557) were transferred to liquid culture medium (by mass percentage, the liquid culture medium consisted of 1.0% peptone, 0.5% sodium chloride, 0.3% beef extract, and 98.2% distilled water) and cultured for 24 hours to obtain Bacillus subtilis subsp. subtilis fermentation seed liquid;

[0123] (3) Preparation of fermentation medium: The fermentation medium comprises the following components by mass percentage: 4% sea rice powder obtained in step (1), 0.5% sodium chloride, 0.1% potassium dihydrogen phosphate, 91.4% distilled water, pH natural (about 7.0);

[0124] (4) Fermentation of sea rice by Bacillus subtilis: Based on the total volume of the fermentation medium obtained in step (3) above, 4% of the Bacillus subtilis fermentation seed liquid obtained in step (2) was inoculated into the above fermentation medium and fermented at 30°C and 160 rpm. After fermentation for 60 h, the fermentation mixture was centrifuged to remove the bacterial cells and obtain the fermentation product.

[0125] Comparative Example 3

[0126] A sea rice powder extract is provided, prepared according to the following steps:

[0127] (1) After crushing the sea rice (commercially available sea red rice, sourced from Zhanjiang, Guangdong) into powder, pass it through a 200-mesh sieve to obtain sea rice powder for later use;

[0128] (2) Combined heating-ultrasound extraction: Take 4% of the sea rice powder obtained in step (1) by weight and mix it with 96% of the extraction solvent (composed of 20% ethanol, 20% glycerol and 60% water by weight). Stir at 60°C for 12 hours, and then sonicate for 60 seconds using an ultrasonic disruptor (ultrasound conditions: 25°C, power: 300W). Centrifuge the extract and collect the supernatant to obtain the extract.

[0129] The performance of the samples obtained from the above embodiments and comparative examples was tested.

[0130] Experimental Example 1

[0131] Determination of lactic acid and inositol hexaphosphate content

[0132] The method for detecting lactic acid content is as follows: Take 15000g of each of the above fermentation products, centrifuge at 4℃ for 10min, and collect the supernatant; determine the lactic acid content of the supernatant of each fermentation product according to the instructions of the lactic acid assay kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0133] The method for detecting inositol hexaphosphate is as follows: Take 15000g of each of the above fermentation products, centrifuge at 4℃ for 10min, and collect the supernatant; determine the inositol hexaphosphate content of the supernatant of each fermentation product according to the instructions of the inositol hexaphosphate content assay kit (purchased from Suzhou Grace Biotechnology Co., Ltd.).

[0134] The release rate of inositol hexaphosphate is calculated using the following formula:

[0135] Inositol hexaphosphate release rate (%) = (Inositol hexaphosphate release amount in supernatant / Inositol hexaphosphate release amount in sea rice) * 100%

[0136] In the above formula, the release of inositol hexaphosphate in the supernatant was determined by the aforementioned method; the inositol hexaphosphate content of sea rice is approximately 983.4 mg / kg (Reference: Lai Shuangding, Long Yu, Chen Yijun, et al. Research progress on nutritional functional components and processing utilization of sea rice [J]. Grains and Oils, 2022, 35(06):13-15+35).

[0137] The test results for each embodiment and comparative example are shown in the table below:

[0138] Table 1

[0139]

[0140] As can be seen from the test results in Table 1 above, the solutions of various embodiments of this application can produce fermentation products that are simultaneously rich in lactic acid and inositol hexaphosphate.

[0141] Comparative Examples 1-4 could only produce either lactic acid or inositol hexaphosphate individually; they could not produce both lactic acid and inositol hexaphosphate simultaneously. This demonstrates that other bacterial strains selected in the comparative examples, conventional physical extraction methods, or the absence of sea rice in the culture medium cannot achieve the technical effects of the embodiments of this application.

[0142] As can be seen, the Bacillus coagulans selected in this embodiment utilizes the nutrients in sea rice for growth and releases the endogenous active ingredients of sea rice, which can greatly increase the content of inositol hexaphosphate in the fermentation broth and promote the production of lactic acid by Bacillus coagulans. This results in the fermentation product being rich in both active ingredients.

[0143] Furthermore, a comparison between Examples 1 and 2, and between Examples 1 and 3, reveals that the inoculum size significantly affects the growth of Bacillus coagulans utilizing nutrients in sea rice and promoting the release of inositol hexaphosphate. In Example 2, when the inoculum size (2%) was lower than that in Example 1 (4%), the increase in the number of Bacillus coagulans was smaller within the same time frame (60h), leading to a decrease in the efficiency of decomposing sea rice and a reduction in the inositol hexaphosphate content. In Example 3, when the inoculum size (8%) was higher than that in Example 1 (4%), the number of Bacillus coagulans was larger, and it could rapidly decompose and consume sea rice within a certain time to release a large amount of inositol hexaphosphate. However, because its quantity far exceeded the capacity of the fermentation system, there was an overabundance of Bacillus coagulans. After the nutrients were depleted, a considerable portion of the inositol hexaphosphate was decomposed and utilized by Bacillus coagulans, ultimately resulting in a reduction in the inositol hexaphosphate content. This suggests that choosing an inoculum size of 2%-8% is more conducive to increasing the inositol hexaphosphate content in the fermentation broth and promoting the production of lactic acid by Bacillus coagulans.

[0144] Furthermore, a comparison between Examples 1 and 4, and between Examples 1 and 5, reveals that the amount of sea rice added to the culture medium significantly affects the growth of Bacillus coagulans using nutrients from sea rice and the promotion of inositol hexaphosphate release. In Example 5 (8%), the proportion of sea rice added was higher than in Example 1 (4%), but this exceeded the decomposition capacity of Bacillus coagulans, therefore the inositol hexaphosphate content did not increase significantly. In Example 4 (2%), the proportion of sea rice added was lower than in Example 1 (4%), and the inositol hexaphosphate content was lower than in Example 1. This indicates that when the amount of sea rice added to the culture medium is 2%-8%, it is more beneficial to increase the inositol hexaphosphate content in the fermentation broth and simultaneously promote lactic acid production by Bacillus coagulans.

[0145] Furthermore, a comparison between Examples 1 and 6, and between Examples 1 and 7, reveals that fermentation time affects the growth of Bacillus coagulans using nutrients from sea rice and promotes the release of inositol hexaphosphate. In Example 6 (48h), the fermentation time was shorter than that of Example 1 (60h), resulting in a certain decrease in lactic acid and inositol hexaphosphate in the fermentation product. This is because the fermentation time of Example 6 (48h) was insufficient compared to Example 1 (60h), leading to a certain degree of reduction in the lactic acid and inositol hexaphosphate content in its fermentation product. In Example 7 (72h), the fermentation time was longer than that of Example 1 (60h), but the lactic acid and inositol hexaphosphate content in the fermentation product were not higher than those in Example 1. This may be because the excessively long fermentation time caused some lactic acid and inositol hexaphosphate to decompose. Therefore, a fermentation time of 48h-72h is more conducive to increasing the inositol hexaphosphate content in the fermentation broth and promoting lactic acid production by Bacillus coagulans.

[0146] Experiment Example 2: Skin Cell Growth and Repair Capacity Test

[0147] This experiment tested the ability of the samples prepared by the comparative examples of each embodiment to promote the growth and repair of human fibroblasts (HDF) in order to evaluate their efficacy in promoting skin wound healing. The fermentation broth of each embodiment was filtered through a 0.22 μm microporous membrane for later use.

[0148] The specific experimental steps for the growth promotion test are as follows:

[0149] After incubating 150 μL of HDF cells at 37°C for 24 h, a high concentration of glucose (35 mmol / L) and fermentation broth (2% concentration) were added, and the cells were incubated at 37°C for another 24 h. Subsequently, 150 μL of CCK-8 solution was added to label live cells. For the sample blank control, 1% PBS buffer was added instead of the fermentation broth. After incubation at 37°C for 30 min, the absorbance was measured at 450 nm.

[0150]

[0151] In the formula:

[0152] A1: Sample well;

[0153] B1: Blank reference well.

[0154] The specific experimental steps for the cell repair test are as follows:

[0155] Place the wound healing four-well insert into a 12-well plate, and seed HDF cells in the logarithmic growth phase into the 12-well plate. Add 110 μL of cell suspension to each well of the four-well insert, with a seeding density of 1*10⁴ cells / well. 5 Cells per well. Incubate at 37°C in a CO2 incubator for 24 hours. Once the cells have largely reached confluence, gently remove the four-well wound healing insert, wash once with PBS, add 110 μL of 0% serum culture medium containing 2% of the samples from each example to each well of the insert, and photograph the cells. Incubate at 37°C in a CO2 incubator until the set observation time. At the set time, observe and measure the width of the scratch under a microscope and photograph the result.

[0156]

[0157] In the formula:

[0158] A1: Scratch width over 24 hours;

[0159] B1: Scratch width at 0 hours.

[0160] The test results of each embodiment and comparative sample are shown in Table 2.

[0161] Table 2. Skin cell growth promotion capacity of different embodiments (2%, v / v)

[0162]

[0163]

[0164] The test results in Table 2 show that:

[0165] Compared with the blank control group, the fermentation products rich in lactic acid and inositol hexaphosphate prepared in each example can effectively improve the survival rate and healing rate of HDF cells, indicating that the fermentation products have a significant ability to promote skin cell growth and repair.

[0166] Compared to the positive control group (10% fetal bovine serum, a nutrient-rich component), the HDF cell survival rate of each embodiment was 80%–97% of that of the positive control group, and the HDF cell healing rate of each embodiment was 32%–99% of that of the positive control group.

[0167] The survival rate and healing rate of HDF cells prepared with fermentation products rich in lactic acid and inositol hexaphosphate in each embodiment were higher than those in each comparative embodiment.

[0168] This demonstrates that the fermentation products of each embodiment have a significant ability to promote skin cell growth and repair.

[0169] Experiment Example 3: Test of Skin Cell Repair Capacity (Part Two)

[0170] Skin glycation is the process by which sugars and proteins combine to form AGEs (advanced glycation end products), leading to problems such as dull, rough skin, aging, and acne. In addition to causing dull, sagging, loose skin and wrinkles, excessively high blood sugar levels can also slow down the healing of acne scars and skin wounds. Therefore, high-glycemic skin models are commonly used to evaluate the effects of raw materials on skin cell growth, in order to clarify their efficacy in promoting wound healing.

[0171] The Wnt / β-catenin pathway plays a crucial role in many biological processes, including cell proliferation, differentiation, and migration. It has been described as a key regulator of skin wound healing; Wnt / β-catenin signaling is upregulated in response to injury and participates in all stages of the healing process. High glucose levels can inhibit wound healing by reducing human fibroblast proliferation through the Wnt signaling pathway.

[0172] This experiment tested the ability of samples prepared in the comparative examples of each embodiment to increase the expression level of growth factor in high-glucose model cells in order to evaluate their ability to promote skin growth. The fermentation broth (or extraction broth) of the samples prepared in the comparative examples of each embodiment was filtered through a 0.22 μm microporous membrane for later use.

[0173] The specific experimental steps are as follows:

[0174] Harvesting healthy human fibroblasts (HDF), at a ratio of 1 × 10⁶ cells per well. 6 To determine the cell density, HDF cells were seeded into 6-well plates, with 2 mL of culture medium added to each well, and incubated at 37°C for 24 h.

[0175] Normal group: cells present, glucose absent, no fermentation broth from the examples and comparative examples; Model group: cells present, glucose (33 mmol / L) present, no fermentation broth from the examples and comparative examples; Intervention group: cells present, fermentation broth from the examples and comparative examples at a final concentration of 2%. The culture medium was changed, and after 2 hours of incubation with the fermentation broth, a glucose solution at a final concentration of 33 mmol / L was added. Cells were harvested after 24 hours of incubation. Total RNA was extracted from cells in each experimental group using a nucleic acid extraction kit and reverse transcribed into cDNA. The sequences and related information of human growth factor Wnt3A and β-catenin were identified. Primers were synthesized by the company, and RT-PCR was performed according to the kit instructions to detect the mRNA expression levels of Wnt3A and β-catenin in each group. －△△C The relative expression levels of growth factors Wnt3A and β-catenin were calculated using the t-method.

[0176] The test results for each embodiment and comparative example are shown in Table 3 below:

[0177] Table 3. Anti-inflammatory and anti-aging capabilities of different embodiments (2%, v / v)

[0178]

[0179]

[0180] The test results in Table 3 show that:

[0181] Compared with the blank control group, the fermentation products rich in lactic acid and inositol hexaphosphate prepared in each example can effectively increase the relative expression levels of Wnt3A and β-catenin, indicating that the fermentation products have the ability to significantly increase the expression levels of growth factors in high-glucose model cells and promote skin growth.

[0182] Compared with the model group, the relative expression levels of Wnt3A and β-catenin in each embodiment were significantly increased, indicating that the fermentation product has a significant effect on improving the inhibition of cell growth by high glucose and promoting skin growth.

[0183] The relative expression levels of Wnt3A and β-catenin in the fermentation products rich in lactic acid and inositol hexaphosphate prepared in each example were higher than those in the comparative examples.

[0184] Experiment Example 4: Skin Cell Anti-inflammatory Capacity Test

[0185] This experiment tested the ability of samples prepared in each embodiment and comparative example to inhibit the expression of inflammatory factors in high-glucose model cells in order to evaluate their ability to promote skin growth. The fermentation broth of each embodiment was filtered through a 0.22 μm microporous membrane for later use.

[0186] The specific experimental steps are as follows:

[0187] Harvesting healthy human fibroblasts (HDF), at a ratio of 1 × 10⁶ cells per well. 6 To determine the cell density, HDF cells were seeded into 6-well plates, with 2 mL of culture medium added to each well, and incubated at 37°C for 24 h.

[0188] Normal group: cells present, LPS absent, no fermentation broth from the examples and comparative examples; Model group: cells present, LPS (10 μg / mL) present, no fermentation broth from the examples and comparative examples; Intervention group: cells present, fermentation broth from the examples and comparative examples at a final concentration of 2%. The culture medium was changed, and after 2 hours of incubation with the fermentation broth, LPS at a final concentration of 10 μg / mL was added. Cells were harvested after 24 hours of incubation. Total RNA was extracted from cells in each experimental group using a nucleic acid extraction kit and reverse transcribed into cDNA. Sequences and related information of human inflammatory factors NLRP3 and IL-18 were identified. Primers were synthesized by the company, and RT-PCR was performed according to the kit instructions to detect the mRNA expression levels of NLRP3 and IL-18 in each group. －△△Ct The relative expression levels of inflammatory factors NLRP3 and IL-18 were calculated using a method.

[0189] Table 4. Anti-inflammatory efficacy of different embodiments (2%, v / v)

[0190]

[0191]

[0192] The test results in Table 4 show that:

[0193] Compared with the blank control group, the fermentation products rich in lactic acid and inositol hexaphosphate prepared in each example can effectively increase the relative expression levels of NLRP3 and IL-18, indicating that the fermentation products have a significant effect on improving the anti-inflammatory ability of skin cells.

[0194] Compared with the model group, the relative expression levels of NLRP3 and IL-18 in each embodiment were significantly reduced, indicating that the fermentation product has a significant effect in improving high glucose-induced cellular inflammation and inhibiting skin inflammation.

[0195] The relative expression levels of NLRP3 and IL-18 in the fermentation products rich in lactic acid and inositol hexaphosphate prepared in each embodiment were lower than those in the comparative examples, indicating that the anti-inflammatory ability of the fermentation products in each embodiment was superior to that in the comparative examples.

[0196] Experiment Example 5: Antibacterial Ability Test

[0197] The minimum inhibitory concentrations (MICs) of the fermentation broths of each example and comparative example against Staphylococcus aureus (purchased from Guangdong Provincial Microbial Culture Collection Center, No. GDMCC 1.174) and Pseudomonas aeruginosa (purchased from Guangdong Provincial Microbial Culture Collection Center, No. GDMCC 1.175) were investigated.

[0198] The specific experimental steps are as follows:

[0199] Single colonies of Staphylococcus aureus were transferred to CAMHB broth medium (purchased from Qingdao Haibo Biotechnology) and cultured for 24-48 hours to obtain a seed culture (OD600 value of approximately 0.8). The seed culture was then diluted with CAMHB broth medium at a volume ratio of 1:1000 for later use.

[0200] Single colonies of Pseudomonas aeruginosa were transferred to CAMHB broth medium and cultured for 24-48 hours to obtain a seed culture (OD600 value of approximately 0.8). The seed culture was then diluted with CAMHB broth medium at a volume ratio of 1:1000 for later use.

[0201] Add 180 μL of *Pseudomonas aeruginosa* or *Staphylococcus aureus* bacterial suspension (diluted 1000-fold) to each well of a 96-well plate, and add 20 μL of the fermentation stock broth from each example and comparative example to achieve final concentrations of 64%, 32%, 16%, 8%, 4%, 2%, 1%, 0.5%, 0.25%, and 0.125%. A negative control group (containing 20 μL of PBS buffer) and a blank control group (containing 200 μL of CAMHB broth) were set up. Incubate at 37°C for 16-20 h. The concentration at which no colonies appear and the plate is clear is the minimum inhibitory concentration (MIC) for that sample.

[0202] The test results for each embodiment and comparative example are shown in Table 5:

[0203] Table 5. Minimum inhibitory concentration (%) of different embodiments

[0204] Example 1 1 4 Example 2 32 32 Example 3 16 16 Example 4 32 32 Example 5 1 4 Example 6 32 32 Example 7 1 4 Comparative Example 4 / / Comparative Example 1 64 64 Comparative Example 2 64 64 Comparative Example 3 64 64

[0205] As shown in Table 5:

[0206] The minimum inhibitory concentrations against Pseudomonas aeruginosa and Staphylococcus aureus in each embodiment were significantly lower than those in each comparative example, indicating that the inhibitory abilities of each embodiment against Pseudomonas aeruginosa and Staphylococcus aureus were superior to those in each comparative example.

[0207] As shown in Experiments 2, 3, 4 and 5, fermentation products rich in both lactic acid and inositol hexaphosphate can effectively enhance the growth and migration rate of skin cells, inhibit inflammatory factors, and suppress the growth of skin pathogens.

[0208] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing inositol hexaphosphate from sea rice through bidirectional fermentation, characterized in that, include: The sea rice grains are crushed to obtain sea rice powder; The Bacillus coagulans seed culture was inoculated into a fermentation medium and fermented to obtain a fermentation mixture; the Bacillus coagulans was purchased from the Guangdong Provincial Microbial Culture Collection Center, with the number GDMCC1.645; The fermentation mixture was centrifuged to remove the bacterial cells, and the fermentation product was obtained; the fermentation product contained Bacillus coagulans lactic acid and inositol hexaphosphate. In the fermentation product, the release rate of inositol hexaphosphate is greater than or equal to 33.5%; The fermentation product contains more than 0.69 g / L of lactic acid from Bacillus coagulans. The step of inoculating the Bacillus coagulans seed culture into the fermentation medium for fermentation includes: Inoculate the Bacillus coagulans seed culture into the fermentation medium and ferment for 48-72 hours. The fermentation medium consists of 2%~8% sea rice powder, 0.1%~1.0% magnesium sulfate, and the remainder is water and the Bacillus coagulans seed solution.

2. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 1, characterized in that, The Bacillus coagulans seed culture incorporated into the fermentation medium accounts for 2% to 8% of the volume of the fermentation medium, by volume percentage.

3. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 1, characterized in that, The amount of sea rice powder added to the fermentation medium is 2% to 8% by mass percentage.

4. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 3, characterized in that, The pH of the fermentation medium is 7.5 to 8.

5.

5. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 1, characterized in that, The preparation of the Bacillus coagulans seed solution includes: Transfer single colonies of Bacillus coagulans to liquid culture medium and culture for 24-48 hours.

6. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 5, characterized in that, The liquid culture medium comprises, by weight percentage: 1.0%~2.0% tryptone, 0.5%~1.5% yeast extract, 0.1%~1.0% magnesium sulfate, and the balance being water.

7. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to claim 6, characterized in that, The pH of the liquid culture medium is 7.5 to 8.

5.

8. The method for preparing inositol hexaphosphate from sea rice by bidirectional fermentation according to any one of claims 1-7, characterized in that, The step of inoculating the Bacillus coagulans seed culture into the fermentation medium for fermentation includes: The Bacillus coagulans seed culture was inoculated into the fermentation medium and fermented at 35℃~38℃ and 230 rpm~250 rpm.

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