Black rice fermented powder, preparation method and application thereof

Abstract

This invention relates to a black rice fermentation powder, its preparation method, and its application, belonging to the field of food fermentation and processing technology. This invention provides a method for preparing black rice fermentation powder, comprising the following steps: (1) crushing black rice and sterilizing it to obtain black rice flour; (2) inoculating the black rice flour with a fermenting agent to obtain a fermentation substrate; (3) mixing the fermentation substrate with water for fermentation and freeze-drying to obtain black rice fermentation powder. This invention uses a specific combination of *Saccharomyces cerevisiae* and *Bacillus amyloliquefaciens* as a fermenting agent to ferment black rice flour. Through experiments, the optimal fermentation parameters were determined. The resulting black rice fermentation powder exhibits significantly higher levels of active ingredients and antioxidant capacity than unfermented and single-strain fermented products, fully verifying the advanced nature, synergy, and high efficiency of this application.

Description

Technical Field

[0001] This invention relates to the field of food fermentation and processing technology, and in particular to a black rice fermentation powder, its preparation method, and its application. Background Technology

[0002] Black rice is a highly nutritious grain resource, rich in protein, vitamins, trace elements, and unique bioactive substances such as anthocyanins, phenolic acids, and flavonoids. These active ingredients endow black rice with excellent antioxidant, anti-inflammatory, and chronic disease prevention benefits, making it an ideal raw material for developing functional foods. Currently, to further improve the content and bioavailability of active ingredients in black rice, physical, chemical, or biological methods are commonly used. Among these, microbial fermentation has attracted much attention due to its mild conditions, environmental friendliness, and ability to produce new flavor compounds and active ingredients. Existing technologies include research on fermenting black rice using single strains such as lactic acid bacteria, molds, or yeasts. However, single-strain fermentation often suffers from limited conversion efficiency, low increases in active ingredient levels, and long fermentation cycles. For example, brewer's yeast fermentation mainly utilizes sugars and may produce ethanol and organic acids, but its ability to degrade complex polysaccharides and fiber is limited; while some Bacillus species possess strong enzyme systems capable of degrading macromolecules and releasing bound phenols, their metabolites and flavor compounds are relatively simple. Furthermore, while traditional chemical extraction methods (such as acid extraction and alcohol extraction) can obtain active ingredients, they suffer from drawbacks such as solvent residue, high energy consumption, and potential damage to natural conformations, and do not fundamentally increase the content of active ingredients in the raw materials themselves. Therefore, developing an efficient and synergistic microbial fermentation technology that leverages the metabolic complementarity of multiple microbial strains to deeply explore the active potential of black rice, significantly increase its functional component content, and shorten the production cycle is of great significance for the high-value utilization of black rice.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a black rice fermentation powder, its preparation method and application, in order to solve the problems of limited fermentation conversion efficiency, low increase in active ingredients and long fermentation cycle of black rice powder in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing black rice fermented powder, comprising the following steps: (1) Grind the black rice into powder and sterilize it for 15-25 minutes to obtain black rice powder; (2) Inoculate the black rice flour with a starter culture to obtain a fermentation substrate; (3) Mix the fermentation substrate with water and ferment for 12-24 hours, then freeze-dry to obtain black rice fermentation powder; The fermentation agent mentioned in step (2) includes Saccharomyces cerevisiae and Bacillus amyloliquefaciens, and the ratio of live Saccharomyces cerevisiae to Bacillus amyloliquefaciens is 3~9:1~7.

[0006] Preferably, the mesh size of the powder in step (1) is 70 to 90 mesh.

[0007] Preferably, the sterilization temperature in step (1) is 75~85℃.

[0008] Preferably, the number of viable bacteria in the fermentation substrate in step (2) is 1×10⁻⁶. 6 ~1×10 8 cfu / g.

[0009] Preferably, the mass-to-volume ratio of the fermentation substrate to water in step (3) is 1g:1~2.5mL.

[0010] Preferably, the fermentation temperature in step (3) is 29~37℃.

[0011] Preferably, the fermentation speed in step (3) is 150~170 rpm.

[0012] This invention provides black rice fermented powder prepared by the aforementioned preparation method.

[0013] This invention provides the application of the aforementioned black rice fermentation powder in the preparation of health foods that enhance immunity.

[0014] This invention provides the application of the aforementioned black rice fermentation powder in the preparation of anti-aging cosmetics.

[0015] The present invention has the following technical effects and advantages: This invention uses a specific ratio of Saccharomyces cerevisiae and Bacillus amyloliquefaciens as a starter culture to ferment black rice flour. Through experiments, the optimal fermentation parameters were determined. The resulting fermented black rice powder showed significantly higher levels of active ingredients and antioxidant capacity compared to unfermented and single-strain fermented products, fully demonstrating the advanced nature, synergy, and high efficiency of the proposed solution. Attached Figure Description

[0016] Figure 1 Results of anthocyanin content determination in black rice fermentation powder prepared with different live bacteria ratios; Figure 2 Results of anthocyanin content determination in black rice fermented powder prepared with different inoculum amounts; Figure 3 Results of anthocyanin content determination in black rice fermentation powder prepared with different material-liquid ratios; Figure 4 Results of anthocyanin content determination in black rice fermented powder prepared at different fermentation temperatures; Figure 5 Results of anthocyanin content determination in black rice fermented powder prepared at different fermentation times. Detailed Implementation

[0017] This invention provides a method for preparing black rice fermented powder, comprising the following steps: (1) Grind the black rice into powder and sterilize it for 15-25 minutes to obtain black rice powder; The sterilization time is preferably 20 minutes; (2) Inoculate the black rice flour with a starter culture to obtain a fermentation substrate; (3) Mix the fermentation substrate with water and ferment for 12-24 hours, then freeze-dry to obtain black rice fermentation powder; The fermentation time is preferably 18 hours; The fermentation agent in step (2) includes Saccharomyces cerevisiae and Bacillus amyloliquefaciens, and the ratio of live bacteria of Saccharomyces cerevisiae to Bacillus amyloliquefaciens is 3~9:1~7, preferably 3:7.

[0018] In this invention, the mesh size of the pulverized material in step (1) is 70-90 mesh, preferably 80 mesh.

[0019] In this invention, the sterilization temperature in step (1) is 75~85℃, preferably 80℃.

[0020] In this invention, the viable cell count in the fermentation substrate in step (2) is 1 × 10⁻⁶. 6 ~1×10 8 cfu / g, preferably 1×10 7 cfu / g.

[0021] In this invention, the mass-to-volume ratio of the fermentation substrate to water in step (3) is 1g:1~2.5mL, preferably 1g:1mL.

[0022] In this invention, the fermentation temperature in step (3) is 29~37℃, preferably 33℃.

[0023] In this invention, the fermentation speed in step (3) is 150~170 rpm, preferably 160 rpm.

[0024] This invention provides black rice fermented powder prepared by the aforementioned preparation method.

[0025] This invention provides the application of the aforementioned black rice fermentation powder in the preparation of health foods that enhance immunity.

[0026] This invention provides the application of the aforementioned black rice fermentation powder in the preparation of anti-aging cosmetics.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] In the examples, the Saccharomyces cerevisiae (ATCC 9763) was purchased from the China Industrial Microbial Culture Collection Center, product code 0699K, and the Bacillus amyloliquefaciens (strain number CCTCC FB 2024255) was purchased from the China Center for Type Culture Collection (Wuhan, China), platform resource number 1542C0001WFB2024255.

[0029] Example 1

[0030] The effect of the inoculation ratio of Saccharomyces cerevisiae and Bacillus amyloliquefaciens in the fermentation agent on the anthocyanin content in black rice fermentation powder

[0031] 1. Preparation of fermentation agent

[0032] (1) Inoculate the Saccharomyces cerevisiae into a culture medium (using water as a solvent, add 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, and 20g of glucose per liter of water, and adjust the pH to 7.0), and culture at 37℃ for 12h. Centrifuge to collect the cells, and resuspend them in sterile physiological saline to obtain a Saccharomyces cerevisiae suspension.

[0033] (2) Bacillus amyloliquefaciens was inoculated into a culture medium (using water as a solvent, adding 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 10g of beef extract, and 20g of glucose per liter of water, and adjusting the pH to 7.0), and cultured at 37℃ for 4 days. The bacterial cells were collected by centrifugation and resuspended in sterile physiological saline to obtain a Bacillus amyloliquefaciens bacterial suspension.

[0034] (3) The yeast suspension and the amyloliquefaciens suspension were mixed in proportions of 1:9, 3:7, 5:5, 7:3 and 9:1 to obtain black rice fermentation powder prepared by fermentation agents with different proportions of live bacteria.

[0035] 2. Preparation of black rice yeast

[0036] (1) Select black rice that is free from mold and insects, grind it to a particle size of 80 mesh, place it in a 150mL conical flask, sterilize it in water at 80℃ for 20min to obtain black rice powder; (2) Add 1×10 to the black rice flour 8 Fermentation substrates were obtained by inoculating the starter culture with an inoculum amount of cfu / g. (3) The fermentation substrate and water were mixed at a mass-volume ratio of 1g:1mL, and fermented for 18h at a temperature of 37℃ and a rotation speed of 160rpm. The black rice fermentation powder prepared by fermentation agents with different live bacteria ratios was obtained by freeze drying.

[0037] 3. Determination of anthocyanin content in black rice fermented powder

[0038] The method for determining anthocyanin content is as follows: Black rice fermentation powder was extracted with 85% ethanol containing 1% HCl by ultrasonication. The supernatant was then mixed with 0.025 mol / L KCl-HCl buffer (pH=1.0) at a volume ratio of 1:9. The UV absorbance was measured at 520 nm and 700 nm, and recorded as A1. 520 A1 700 The supernatant was mixed with 0.4 mol / L NaAc-HAc buffer (pH=4.5) at a volume ratio of 1:9, and the UV absorbance was measured at 520 nm and 700 nm, respectively, and recorded as A2. 520 A2 700 The blank control group consisted of corresponding buffer solutions. Anthocyanin content (calculated as cyanidin-3-glucoside) was calculated using the following formula:

[0039] in the formula MW This indicates the molecular weight of cyanidin-3-glucoside, specifically 449.2 g / mol; DF represents the sample dilution factor. ε It is the extinction coefficient of cyanidin-3-glucoside, with a specific value of 26900 L / (mol·cm); Ι The optical path length of the cuvette is 1 cm in this application.

[0040] The anthocyanin content was determined using the above method. The results of anthocyanin content determination in black rice fermented powder prepared with different ratios of live bacteria inoculants are as follows: Figure 1 As shown.

[0041] according to Figure 1 It can be seen that the anthocyanin content first increases and then decreases with the change in the ratio of microbial strains. When the ratio of viable cells of *Saccharomyces cerevisiae* to *Bacillus amyloliquefaciens* is 3:7, the anthocyanin content of fermented black rice reaches its peak (24.23%), which is significantly higher than that of other ratio groups. P <0.05). This indicates that the metabolic synergy between the two bacteria is strongest at this ratio, therefore the optimal live bacteria ratio of Saccharomyces cerevisiae to Bacillus amyloliquefaciens in the fermentation agent is determined to be 3:7.

[0042] Example 2

[0043] Effect of different inoculum amounts on anthocyanin content in black rice yeast

[0044] Black rice flour was fermented using the method described in "2. Preparation of Black Rice Fermentation Powder" in Example 1. The difference was that the fermenting agent used was the same as in Example 1, with a ratio of 3:7 between *Saccharomyces cerevisiae* and *Bacillus amyloliquefaciens* viable cells. Furthermore, different inoculation amounts of fermenting agent were used, resulting in five different inoculation amounts, each producing a viable cell count of 1×10⁻⁶ cells / mL in the fermentation substrate. 4 cfu / g, 1×10 5 cfu / g, 1×10 6 cfu / g, 1×10 7 cfu / g and 1×10 8 CFU / g was used to obtain black rice fermented powder prepared with different inoculum amounts. The anthocyanin content was determined using the method described in Example 1, "3. Determination of anthocyanin content in black rice fermented powder". The results of anthocyanin content determination in black rice fermented powder prepared with different inoculum amounts are as follows: Figure 2 As shown.

[0045] according to Figure 2 It can be seen that the anthocyanin content increases significantly with the increase of the inoculum size. When the inoculum size of the fermentation agent is 1×10⁻⁶, the content increases significantly. 8 At a concentration of cfu / g, anthocyanin content was highest, and further increasing the inoculum amount did not significantly increase it; in fact, it might inhibit metabolism due to excessive nutrient competition. Therefore, the optimal inoculum amount was determined to be 1×10⁻⁶. 8 cfu / g.

[0046] Example 3

[0047] Effect of different material-to-liquid ratios on anthocyanin content in black rice yeast

[0048] Black rice flour was fermented using the method described in "2. Preparation of Black Rice Fermentation Powder" of Example 1. The difference was that the fermenting agent used was the same as in Example 1, with a ratio of 3:7 between *Saccharomyces cerevisiae* and *Bacillus amyloliquefaciens* viable cells. Five different material-to-liquid ratios (g:mL) of fermentation substrate to water were also set: 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5, resulting in black rice fermentation powder prepared with different material-to-liquid ratios. The anthocyanin content was determined using the method described in "3. Determination of Anthocyanin Content in Black Rice Fermentation Powder" of Example 1. The anthocyanin content determination results for the black rice fermentation powder prepared with different material-to-liquid ratios are as follows: Figure 3 As shown.

[0049] according to Figure 3It is evident that moisture significantly affects fermentation efficiency. The highest anthocyanin content was observed when the ratio of fermentation substrate to water was 1 g:1 mL. Insufficient moisture (1 g:0.5 mL) limited microbial growth and substance diffusion; excessive moisture (1 g:2 mL, 1 g:2.5 mL) may cause the system to become viscous, reduce dissolved oxygen, and dilute the substrate concentration, all of which are detrimental to the biotransformation and accumulation of bioactive substances. Therefore, the optimal ratio of fermentation substrate to water was determined to be 1 g:1 mL.

[0050] Example 4

[0051] Effect of different fermentation temperatures on anthocyanin content in black rice yeast

[0052] Black rice flour was fermented using the method described in "2. Preparation of Black Rice Fermentation Powder" of Example 1, except that the fermenting agent used was a 3:7 ratio of *Saccharomyces cerevisiae* to *Bacillus amyloliquefaciens*, as described in Example 1. Five different fermentation temperatures were set: 25°C, 29°C, 33°C, 37°C, and 41°C, resulting in black rice fermentation powder prepared at different fermentation temperatures. The anthocyanin content was determined using the method described in "3. Determination of Anthocyanin Content in Black Rice Fermentation Powder" of Example 1. The anthocyanin content determination results for the black rice fermentation powder prepared at different fermentation temperatures are as follows: Figure 4 As shown.

[0053] according to Figure 4 It is evident that fermentation temperature has a decisive influence on cell activity and enzymatic reaction rate. Fermentation at 29℃ resulted in a product with significantly higher anthocyanin content than those at other temperatures. P <0.05). Although 37℃ is a common optimal growth temperature for microorganisms, 29℃ is more favorable for the synthesis of secondary metabolites in this specific mixed-culture system in black rice substrate. Temperatures that are too high (41℃) or too low (25℃) will inhibit metabolic activity. Therefore, the optimal fermentation temperature was finally determined to be 29℃.

[0054] Example 5

[0055] Effect of different fermentation times on anthocyanin content in black rice baking powder

[0056] Black rice flour was fermented using the method described in "2. Preparation of Black Rice Fermentation Powder" of Example 1, except that the fermenting agent used was a 3:7 ratio of *Saccharomyces cerevisiae* to *Bacillus amyloliquefaciens*, as described in Example 1. The fermentation temperature was set to 29°C, and six different fermentation times were set: 12h, 24h, 48h, 72h, 96h, and 120h. Black rice fermentation powder was prepared with different fermentation times. The anthocyanin content was determined using the method described in "3. Determination of Anthocyanin Content in Black Rice Fermentation Powder" of Example 1. The anthocyanin content determination results for black rice fermentation powder prepared with different fermentation times are as follows: Figure 5 As shown.

[0057] according to Figure 5 It was found that the anthocyanin content decreased with increasing fermentation time, reaching a peak at 12 hours and then declining. This indicates that 12 hours is the highly efficient period for the mixed-culture system to metabolize and synthesize active substances. Extending the fermentation time to 120 hours may lead to a decline due to nutrient depletion or accumulation of metabolites, which would not be beneficial for improving the target product and would reduce production efficiency. Therefore, the optimal fermentation time was determined to be 12 hours.

[0058] Example 6

[0059] Based on Examples 1 to 5, the optimal preparation method for black rice yeast powder is as follows: (1) Select black rice that is free from mold and insects, grind it to a particle size of 80 mesh, place it in a 150mL conical flask, sterilize it in water at 80℃ for 20min to obtain black rice powder; (2) Add 1×10 to the black rice flour 8 The fermentation substrate was obtained by inoculating the starter culture (with a live cell ratio of 3:7 for Saccharomyces cerevisiae and Bacillus amyloliquefaciens) at an inoculum amount of cfu / g. (3) The fermentation substrate and water were mixed at a mass ratio of 1g:1mL, fermented at 29℃ and 160rpm for 12h, and then freeze-dried to obtain black rice fermentation powder.

[0060] Comparative Example 1

[0061] Select black rice free from mold and insect infestation, grind it to a particle size of 80 mesh, place it in a 150mL conical flask, and sterilize it in water at 80℃ for 20 minutes to obtain black rice powder.

[0062] Comparative Example 2

[0063] The preparation method is the same as in Example 6, except that only Saccharomyces cerevisiae is used as the starter culture to prepare black rice fermented powder.

[0064] Comparative Example 3

[0065] The preparation method is the same as in Example 6, except that only Bacillus amyloliquefaciens is used as a starter culture to prepare black rice fermented powder.

[0066] Experimental Example

[0067] Determining the active ingredients and antioxidant activity in different products

[0068] (1) Determination of anthocyanin content

[0069] The anthocyanin content in the products prepared in Example 6, Comparative Example 1, Comparative Example 2, and Comparative Example 3 was determined using the method described in Example 1, "3. Determination of anthocyanin content in black rice fermented powder".

[0070] (2) Determination of total phenols, total flavonoids, DPPH, ABTS free radical scavenging capacity and total reducing power

[0071] The products prepared in Example 6, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were mixed with 60% ethanol at a ratio of 1g:30mL. The mixtures were extracted for 30min in an ultrasonic environment at 360W and 29℃, and then centrifuged for 5min at 8000rpm. The supernatant was collected for the determination of total phenols, total flavonoids, DPPH, ABTS free radical scavenging ability, and total reducing power (FRAP method).

[0072] The results of the determination of active ingredients and antioxidant activity in different products are shown in Table 1. Different letters in the table indicate significant differences.

[0073] Table 1. Results of determination of active ingredients and antioxidant activity in different products.

[0074] As shown in Table 1, the black rice fermented powder prepared in Example 6 exhibited significant advantages in all indicators. Analysis revealed that the black rice fermented powder prepared in Example 6 had a much higher enrichment effect on active ingredients than single-strain fermentation (Comparative Examples 2 and 3) and the unfermented group (Comparative Example 1). Specifically, the anthocyanin content was 172.78% higher than that of the black rice powder in Comparative Example 1 and 141.80% higher than that in Comparative Example 3. Correspondingly, its antioxidant capacity was also significantly enhanced, demonstrating that mixed-strain fermentation produced a synergistic effect of "1+1>2".

[0075] As can be seen from the above embodiments, the present invention provides a black rice fermentation powder, its preparation method, and its application. The present invention uses a specific combination of *Saccharomyces cerevisiae* and *Bacillus amyloliquefaciens* as a fermenting agent to ferment black rice flour. Optimal fermentation parameters were determined through experiments, and the resulting black rice fermentation powder exhibited significantly higher levels of active ingredients and antioxidant capacity than unfermented and single-strain fermented products, fully verifying the advanced nature, synergistic effect, and high efficiency of the proposed solution.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing black rice yeast powder, characterized in that, Includes the following steps: (1) Grind the black rice into powder and sterilize it for 15-25 minutes to obtain black rice powder; (2) Inoculate the black rice flour with a starter culture to obtain a fermentation substrate; (3) Mix the fermentation substrate with water and ferment for 12-24 hours, then freeze-dry to obtain black rice fermentation powder; The fermentation agent mentioned in step (2) includes Saccharomyces cerevisiae and Bacillus amyloliquefaciens, and the ratio of live Saccharomyces cerevisiae to Bacillus amyloliquefaciens is 3~9:1~7.

2. The preparation method according to claim 1, characterized in that, The mesh size of the powder to be pulverized in step (1) is 70~90 mesh.

3. The preparation method according to claim 1, characterized in that, The sterilization temperature in step (1) is 75~85℃.

4. The preparation method according to claim 1, characterized in that, The viable cell count in the fermentation substrate described in step (2) is 1 × 10⁻⁶. 6 ~1×10 8 cfu / g.

5. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the fermentation substrate to water in step (3) is 1g:1~2.5mL.

6. The preparation method according to claim 1, characterized in that, The fermentation temperature in step (3) is 29~37℃.

7. The preparation method according to claim 1, characterized in that, The fermentation speed in step (3) is 150~170 rpm.

8. Black rice fermented powder prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the black rice fermentation powder according to claim 8 in the preparation of health food that enhances immunity.

10. The use of the black rice fermentation powder according to claim 8 in the preparation of anti-aging cosmetics.

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