A solid state fermentation process for improving the antioxidant activity of broken rice by using phellinus baumii

Abstract

The application discloses a solid fermentation process for improving the antioxidant activity of broken rice by using phellinus baumii, and belongs to the field of bioengineering, wherein the broken rice is mixed with a culture solution at a ratio of 6 g / mL, sterilized, inoculated with phellinus baumii, and then fermented at 34 DEG C, with uniform mixing once a day, once addition of the culture solution after 5 days, and fermentation for 12 days; the culture solution comprises 20 g / L maltose, 10 g / L peptone and 1.2 g / L magnesium sulfate. The broken rice fermented by phellinus baumii can effectively improve the antioxidant activity of the broken rice. After the broken rice is fermented by phellinus baumii, the total antioxidant capacity of the broken rice is improved by 6.54 times, the DPPH free radical scavenging capacity is improved by 2.08 times, the catalase is improved by 2.06 times, and the superoxide dismutase activity is improved by 11.81 times.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a solid-state fermentation process that utilizes Phellinus linteus to enhance the antioxidant activity of broken rice. Background Technology

[0002] The rice processing method generates a series of byproducts, such as rice husks, broken rice, rice bran, and straw. Broken rice, due to its low market value, is often directly added to livestock feed or processed to replace part of the energy feed, resulting in significant nutrient waste. Improving and enhancing the nutritional value of rice through fermentation technology is an effective approach. Solid-state fermentation (SSF) is a type of fermentation technology that utilizes microorganisms to ferment insoluble substrates on a solid matrix. The fermented material both improves the rice's quality and serves as a source of nutrients. SSF technology is closely related to our lives; common household products such as soy sauce and vinegar are produced through fermentation. In recent years, the method of fermenting grains using SSF technology has been widely adopted because it maximizes the added value of the product. In 2018, Wang et al. reported that after solid-state fermentation of grains using Bacillus subtilis, the protein content in the fermentation product increased by 11.29% compared to the unfermented original grains. In the same year, Xu et al. studied the solid-state fermentation of seven grains—wheat, rice, sorghum, oats, corn, millet, and buckwheat—using edible fungi Agaricus bisporus, Phytosporum aviculare, and Pterocarya stenoptera, respectively. They found that the total phenol content of the fermented grains increased to different degrees depending on the fermentation strain, with different grains showing varying degrees of increase compared to the original grains, the highest being 1.6 times.

[0003] Phellinus linteus is a large fungus that is both edible and has health and pharmacological effects. Its fruiting body, mycelium and fermentation broth contain a variety of active ingredients such as Phellinus linteus polysaccharides, total triterpenes and flavonoids. It has significant antioxidant activity as well as anti-inflammatory, immune-enhancing, blood sugar-lowering, tumor cell metastasis-inhibiting and anti-tumor effects. It is a good natural raw material for scavenging free radicals.

[0004] Gao Jiangeng's 2023 study showed that *Sanghuang* fungus significantly increased the total phenolic content in most grains (rice, sorghum, wheat, corn, etc.), with a maximum increase of 1.62 times. Therefore, organically combining *Sanghuang* fungus with broken rice and using its biotransformation can not only improve the utilization rate and nutritional value of broken rice, but also enhance its functional components. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to use Phellinus linteus to improve the antioxidant activity of broken rice.

[0006] The technical solution of the present invention is as follows: a solid-state fermentation process for improving the antioxidant activity of broken rice using Phellinus linteus. Broken rice and culture medium are mixed at a ratio of 6 g / mL, sterilized, and then Phellinus linteus is inoculated. Fermentation is carried out at 34°C, with mixing once a day. After 5 days, culture medium is added again, and fermentation is carried out for 12 days. The culture medium consists of 20 g / L maltose, 10 g / L peptone, and 1.2 g / L magnesium sulfate.

[0007] Furthermore, the *Sanghuang* is activated by the following method: *Sanghuang* is inoculated onto potato dextrose agar plates and cultured in a 27 ℃ incubator for 7 days. Fresh colonies are then picked and transferred to new PDA plates for purification and culture for 10 days.

[0008] Furthermore, the amount of *Sanghuang* inoculated is 9 *Sanghuang* fungus cakes prepared according to claim 2 for every 60g of broken rice, and the fungus cakes have a diameter of 6mm.

[0009] Furthermore, the amount of culture medium added is 23 ml per 60 g of broken rice.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] Fermenting broken rice with Phellinus linteus effectively enhances its antioxidant activity. After fermentation with Phellinus linteus, the total antioxidant capacity of broken rice increased by 6.54 times, the DPPH free radical scavenging capacity increased by 2.08 times, the catalase activity increased by 2.06 times, and the superoxide dismutase activity increased by 11.81 times. Attached Figure Description

[0012] Figure 1 This is a 3D surface plot of the response surface to temperature and inoculum amount.

[0013] Figure 2 The 3D surface plot of the response surface for temperature and sample loading amount.

[0014] Figure 3 This is a 3D surface plot of the temperature and time response surface.

[0015] Figure 4 The response surface 3D surface plots for inoculation amount and sample loading amount.

[0016] Figure 5 This is a 3D surface plot of the inoculation volume and time response surface.

[0017] Figure 6 This is a 3D surface plot of the sample loading and time response surface. Detailed Implementation

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0019] Example 1: Optimization of conditions for solid-state fermentation of broken rice using Sanghuang (a type of medicinal mushroom) through single-factor experiments

[0020] 1. Materials and Methods

[0021] 1.1 Test strains and broken rice

[0022] The fungal strain of *Sanghuangporus vaninii*, with accession number CGMCC5.891, was purchased from the China General Microbiological Culture Collection Center; the broken rice was provided by Guizhou Tianbao Rice Industry Co., Ltd.

[0023] 1.2 Optimization of Solid-State Fermentation Conditions for Sanghuang Rice

[0024] 1.2.1 Cell activation: Sanghuang fungus was inoculated onto potato dextrose agar (PDA) plates and incubated at 27°C for 7 days. Fresh colonies were picked and transferred to new PDA plates for purification and culture for 10 days. Then, 6 mm diameter mycelial cakes were taken using a punch for later use.

[0025] 1.2.2 Fermentation conditions optimization: Single-factor experiments were conducted to optimize and screen the fermentation conditions (temperature, inoculum amount, sample loading amount and inoculation time) of Sanghuang solid-state fermentation of broken rice.

[0026] Using color difference as an evaluation index (the trend of color difference can indirectly reflect the degree of fungal fermentation; the higher the degree of fermentation, the darker the color and the lower the color difference value), single-factor experiments were conducted based on natural pH, 6 inoculum cakes, temperature 28℃, and time 12d to study the effects of temperature, inoculation time, inoculation amount, and sample loading amount on the color difference value of broken rice in solid-state fermentation of Sanghuang.

[0027] Culture medium preparation: Prepare a solid-state fermentation culture medium of Sanghuang rice using 20 g / L maltose, 10 g / L peptone, and 1.2 g / L magnesium sulfate.

[0028] Add 10 mL of culture medium to 60 g of broken rice, mix well, and then add it to a culture bag (12 cm wide × 24 cm long × 5 cm thick). Sterilize under high pressure for later use.

[0029] The temperature settings are: 25℃, 28℃, 31℃, 34℃, 37℃;

[0030] The inoculation amounts were set as follows: 0 mushroom cakes, 3 mushroom cakes, 6 mushroom cakes, 9 mushroom cakes, and 12 mushroom cakes.

[0031] The time periods are set to: 5 days, 8 days, 11 days, 14 days, and 17 days.

[0032] The sample loading volumes were set to: 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL.

[0033] After inoculating the mycelium cake according to the conditions, mix it well and mix it once a day. After 5 days of inoculation, add the sterile culture medium set for the sample volume and continue to incubate, mixing it well every day. After incubating for the set time, take it out, dry it at a low temperature of 40℃, grind it with a grinder, sieve it (35 mesh), and test its color difference with a colorimeter. The determination of color difference refers to GB / T 7921-2008 "Uniform Color Space and Color Difference Formula".

[0034] 2 Results

[0035] 2.1 Effect of fermentation time on the color difference value of broken rice from solid-state fermentation of Sanghuang

[0036] Using color difference as the evaluation index, different time gradients were set to study the effect of fermentation time on the color difference of broken rice in solid-state fermentation of Sanghuang. The darker the color of the broken rice, the lower the color difference value and the higher the degree of fermentation. The results are shown in Table 1. As the fermentation time increases, the color difference value first decreases, reaching its lowest value at 14 days of fermentation, and then increases again.

[0037] Table 1. Effect of fermentation time on color difference of broken rice from solid-state fermentation of Sanghuang.

[0038]

[0039] 2.2 Effect of inoculum amount on color difference of fermented rice from Phellinus linteus

[0040] Using color difference as the evaluation index, different inoculation amounts were set to study the effect of inoculation amount on the color difference of broken rice in solid-state fermentation of Sanghuang. The results are shown in Table 2. As the fermentation inoculation amount increases, the color difference value first decreases, and the color difference value is the lowest when 9 mycelium cakes are inoculated, and then increases again.

[0041] Table 2. Effect of inoculum amount on color difference of fermented rice from Phellinus linteus.

[0042]

[0043] 2.3 Effect of Temperature on Color Difference of Fermented Rice from Sanghuang

[0044] Using color difference as the evaluation index, different temperature gradients were set to study the effect of fermentation temperature on the color difference of solid-state fermented rice of Sanghuang. The results are shown in Table 3. As the fermentation temperature increases, the color of solid-state fermented rice of Sanghuang changes to a certain extent. When the fermentation temperature is 34℃, the color difference value of the rice is the lowest.

[0045] Table 3. Effect of temperature on color difference of fermented rice from Sanghuang mushroom.

[0046]

[0047] 2.4 Effect of Sample Loading Amount on Color Difference of Fermented Sanghuang Broken Rice

[0048] Using color difference as the evaluation index, different sample loading amounts were set to study the effect of sample loading amount on the color difference of solid-state fermented rice of Sanghuang. The results are shown in Table 4. As the sample loading amount increases, the color of solid-state fermented rice of Sanghuang changes. When the sample loading amount is 20 mL, the color difference value is the lowest.

[0049] Table 4. Effect of sample loading amount on color difference of fermented rice from Sanghuang.

[0050]

[0051] In summary, the optimal inoculum size for solid-state fermentation of Sanghuang rice is 9 mycelial cakes, a sample loading volume of 20 mL of liquid culture medium, a fermentation temperature of 34℃, and a fermentation time of 14 days.

[0052] Example 2 Response Surface Optimization of Solid-State Fermented Broken Rice from Sanghuang

[0053] 1 Method

[0054] Based on the results of the optimization project for solid-state fermentation of broken rice using *Sanghuang* fungus, a four-factor, three-level experiment was conducted. Response surface methodology was implemented using Design-Expert 13 software, and the Box-Benhnken method was employed to determine the optimal formulation of the fermented broken rice substrate. The experiment was set up with three replicates. The factors and levels of the response surface methodology are shown in the table below. After cultivation, the culture medium was ground into powder, and the color difference was measured.

[0055] Single-factor experiments were conducted to determine the inoculation amount, sample loading amount, fermentation temperature, and time for fermenting broken rice with Sanghuang. The inoculation amount, sample loading amount, fermentation temperature, and time were used as independent variables (X), and three levels were designed for each factor. The codes were (-1, 0, 1) and the color difference value was used as the response index (Y). Response surface optimization was performed to obtain 29 experimental schemes, as shown in Table 6.

[0056] Table 5 Factors and levels in response surface methodology

[0057]

[0058] Table 6. Four-Factor Three-Level Experiment

[0059]

[0060] 2 Results

[0061] Based on the experimental results of color difference values ​​in Table 7, the results were fitted using Design-Expert 13 software, yielding the following regression equation: Color difference value Y = 2720.01 - 137.27A - 5.34B - 22.56C - 22.58D - 0.27AB + 0.30AC + 0.35AD + 0.05BC - 0.20BD - 0.18CD + 1.95A2 +0.90B 2 +0.34C 2 +0.55D 2 In the color difference model, P < 0.0001, indicating the model is highly significant; the model correlation coefficient R0 2 =0.9332, the model fit is good; the p-value of the model lack of fit term is 0.0549, the difference is not significant, and the model is consistent with the actual situation.

[0062] Table 7. Analysis of Variance of Response Surface Experiment Regression Model

[0063]

[0064] Note: In the table, ** indicates extremely significant difference (P<0.01), and * indicates significant difference (P<0.05).

[0065] Draw a response surface diagram of color difference values ​​using Design-Expert 13 software. Figures 1-6 The results of the response surface methodology were visualized and analyzed. The 3D response surface plot shows that the parabolic graph of the color difference equations opens upwards, and all equations have minimum values.

[0066] Response surface methodology analysis revealed that the optimal fermentation temperature for solid-state fermentation of Sanghuang fermented rice was 34.15℃, the inoculum size was 8.68, the sample loading amount was 23.23 mL, and the fermentation time was 11.95 days. At this time, the predicted color difference value was 43.55.

[0067] To verify the reliability of the results obtained by the response surface methodology, the factors determined by the response surface methodology were combined with actual operation to determine the fermentation temperature as 34℃, the inoculum size as 9, the sample loading amount as 23mL, the fermentation time as 12d, and the measured color difference value as 41.68, which is close to the model prediction value, indicating that the response surface methodology can be applied to actual prediction.

[0068] Example 3: Fermentation of broken rice with Phellinus linteus to enhance the antioxidant activity of broken rice

[0069] 1. Method

[0070] The total antioxidant activity (T-AOC) of broken rice was determined using a Total Antioxidant Capacity (T-AOC)-FRAP assay kit, the DPPH free radical scavenging capacity of broken rice was determined using a DPPH free radical scavenging assay kit, the catalase (CAT) activity of broken rice was determined using a catalase (CAT) assay kit, and the superoxide dismutase (SOD) activity of broken rice was determined using a superoxide dismutase (SOD) assay kit.

[0071] 2. Antioxidant activity of fermented rice containing Phellinus linteus

[0072] Fermenting broken rice with Phellinus linteus effectively enhances its antioxidant activity. After fermentation with Phellinus linteus, the total antioxidant capacity of broken rice increased by 6.54 times, the DPPH free radical scavenging capacity increased by 2.08 times, the catalase activity increased by 2.06 times, and the superoxide dismutase activity increased by 11.81 times.

[0073] Table 8 Changes in the antioxidant activity of fermented rice containing Sanghuang mushroom

[0074]

[0075] Note: control refers to untreated broken rice, and SH refers to fermented broken rice.

Claims

1. A solid-state fermentation process for enhancing the antioxidant activity of broken rice using Phellinus linteus, characterized in that, Broken rice and culture medium were mixed at a ratio of 6 g / mL, sterilized, and then inoculated with Phellinus linteus. Fermentation was carried out at 34℃, with the mixture being stirred once a day. After 5 days, culture medium was added again, and fermentation was carried out for 12 days. The culture medium consisted of 20 g / L maltose, 10 g / L peptone, and 1.2 g / L magnesium sulfate.

2. The solid-state fermentation process according to claim 1, characterized in that, The *Sanghuang* fungus was activated by the following method: *Sanghuang* fungus was inoculated onto potato dextrose agar plates and cultured in a 27 ℃ incubator for 7 days. Fresh colonies were then picked and transferred to new PDA plates for purification and culture for 10 days.

3. The solid-state fermentation process according to claim 1, characterized in that, The amount of *Sanghuang* inoculated is 9 *Sanghuang* fungus cakes prepared according to claim 2, with a diameter of 6 mm, per 60g of broken rice.

4. The solid-state fermentation process according to claim 1, characterized in that, The amount of culture medium to be added is 23 ml per 60 g of broken rice.

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