Culture medium suitable for high yield of flavone from Wanning phellinus igniarius fermented broken rice

Abstract

A preparation method of the culture medium comprises the following steps: weighing 5g of broken rice flour, putting the weighed broken rice flour into a 250 mL triangular flask, adding 2.0 g of maltose, 1.0 g of peptone and 0.12 g of magnesium sulfate, inoculating five bacterial cakes activated by phellinus igniarius, and carrying out shake-flask culture at the speed of 180 r / min and the temperature of 27 DEG C for 10 days, so as to obtain the culture medium. The total flavone content of the broken rice fermented by the culture medium prepared by the invention is 1.1430 mg / mL, which is higher than that of the broken rice fermented by a basic culture medium; and the dry weight of the thallus is 1.7227 g, which is higher than 0.1089 mg / mL of that of the fermentation of a basic culture medium. According to the method, a suitable substrate of phellinus igniarius fermented broken rice is screened out, an effective culture medium formula is obtained, the phellinus igniarius is suitable for growing on the culture medium containing the broken rice, and the total amount of flavone can be increased. The utilization rate of the broken rice is increased, meanwhile, the broken rice is endowed with richer nutritional value, the phellinus igniarius can have higher flavone content, and the broken rice and the phellinus igniarius supplement each other to achieve a synergistic effect in the whole invention concept.

Description

Technical Field

[0001] The invention belongs to the technical field of multifunctional fungus grain preparation, and specifically discloses a culture medium suitable for fermenting broken rice with Phellinus igniarius to produce high flavonoids. Background Art

[0002] Phellinus igniarius is a large fungus that is both edible and has both health and pharmacological benefits. Its fruiting bodies, mycelium, and fermentation broth all contain a variety of active ingredients, including mulberry igniarius 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, making it a good natural raw material for scavenging free radicals. Phellinus igniarius belongs to the genus Moriformis, family Moriformis, order Agaricales, order Coriolusales, family Coriolusaceae, phylum Basidiomycetes. It is described in Shennong's Herbal Classic and Compendium of Materia Medica as "benefiting the five internal organs, promoting digestion, and removing toxins." Solid-state fermentation of grains using mulberry igniarius as a fermentation strain has been reported to effectively increase the nutritional value and active ingredient content of grains. In a 2023 study, Gao Jiangeng demonstrated that Weigela mulberry igniarius significantly increased the total phenolic content of most raw grains (rice, sorghum, wheat, corn, etc.), with the highest increase being 1.62 times.

[0003] Rice is an essential food in our daily lives. The rice we consume is generally refined, resulting in significant loss of nutrients and the production of large quantities of broken rice. As a byproduct of rice processing, broken rice has low value and a poor market. However, research has shown that fermentation technology can enhance the nutritional content of rice and other products, thereby increasing their added value. Research reports indicate that mulberry igneous plants have a long history of treating arthritis. The Tang Dynasty's "Newly Revised Materia Medica" records that "the golden mulberry ear is a drink for treating arthritis." The Song Dynasty's "Shenghui Fang" and the Qing Dynasty's "Yang's Prescriptions" also mention mulberry igneous plants treating arthritis. Modern research has found that 0.05 g·mL⁻¹ of mulberry igneous extract significantly inhibits the expression of the XOD gene, a key enzyme in uric acid metabolism, in L02 cells. An ethanol extract of mulberry igneous plants can improve renal tubular inflammatory infiltration and protect the kidneys. Low doses of polysaccharides from mulberry igneous fermentation products can significantly inhibit hepatic xanthine oxidase activity. Therefore, total flavonoids from mulberry igneous plants have important anti-inflammatory and uric acid-lowering effects in vitro. By utilizing fermentation technology, using mulberry ignia as the fungus and broken rice as the matrix, we can not only increase the added value of broken rice, but also increase the yield of mulberry ignia flavonoids.

[0004] Since Phellinus igniarius grows differently on different culture media and produces different flavonoids in its secondary metabolites, screening for a suitable substrate for fermenting Phellinus igniarius is of great significance. Therefore, a culture medium formula suitable for high flavonoid production from fermented Phellinus igniarius is urgently needed in the industry. Summary of the Invention

[0005] Based on the above analysis, the present invention screened out the suitable substrate for the fermentation of Phellinus igniarius with Broken Rice, and obtained an effective culture medium formula, which is not only suitable for the growth of Phellinus igniarius on the culture medium containing Broken Rice, but also can increase the total amount of flavonoids.

[0006] In order to achieve the above technical effects, the present invention is implemented by adopting the following technical means:

[0007] A culture medium suitable for fermenting broken rice with Phellinus igniarius to produce high flavonoids is prepared by the following method:

[0008] Weigh 5 g of broken rice flour into a 250 mL Erlenmeyer flask, add 2.0 g of maltose, 1.0 g of peptone and 0.12 g of magnesium sulfate, inoculate 5 bacterial cakes, and culture in a shake flask at 180 rpm and 27°C for 10 days.

[0009] Furthermore, the particle size of the broken rice flour is 35 meshes.

[0010] Furthermore, the mushroom cake is prepared by the following method:

[0011] Inoculate a potato dextrose agar (PDA) plate with Phellinus igniarius and incubate in a 27°C incubator for 7 days. Fresh colonies were transferred to new PDA plates for purification and culture for 10 days. A 6 mm diameter bacterial cake was then obtained using a microporator for later use. Phellinus igniarius was purchased from the China Center for Microbiological Collection, with the accession number CGMCC5.891.

[0012] The broken rice fermented with the culture medium prepared by the present invention has a total flavonoid content of 1.1430 mg / mL, which is higher than that of the broken rice fermented with the basic culture medium; the dry weight of the bacteria is 1.7227 g, which is higher than that of the broken rice fermented with the basic culture medium (0.1089 mg / mL).

[0013] The beneficial effects of the present invention are:

[0014] The present invention screens for a suitable substrate for fermenting Phellinus igniarius with Broken Rice, obtaining an effective culture medium formula that is not only suitable for the growth of Phellinus igniarius on a culture medium containing Broken Rice, but also increases the total amount of flavonoids. This not only improves the utilization rate of Broken Rice, but also provides Broken Rice with a richer nutritional value and also increases the flavonoid content of Phellinus igniarius. The two complement each other and play a synergistic role in the overall inventive concept. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The total flavonoid content and dry weight of the three strains of Phellinus igniarius in the fermentation filtrate after fermenting broken rice;

[0016] Figure 2 The effect of different carbon sources on the content of total flavonoids;

[0017] Figure 3 The effect of different carbon sources on the dry weight of bacteria;

[0018] Figure 4 The effect of different nitrogen sources on the content of total flavonoids;

[0019] Figure 5 The effect of different nitrogen sources on the dry weight of bacteria;

[0020] Figure 6 The effect of different inorganic salts on the content of total flavonoids;

[0021] Figure 7 The effect of different inorganic salts on the dry weight of bacteria. DETAILED DESCRIPTION

[0022] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0023] References to "embodiments" herein mean that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the term "embodiment" in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or relevance to other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the various technical features mentioned in the embodiments can be combined in any manner to form a corresponding implementable technical solution.

[0024] Example 1

[0025] This example is the screening of Phellinus igniarius strains with high flavonoid production:

[0026] (I) Bacterial Activation: Three strains of Phellinus linteus were selected: Phellinus linteus (number A), Phellinus linteus (number B), and Phellinus linteus (number C); A and C were purchased from the China National Center for Microbiological Collection with accession numbers CGMCC5.1262 and CGMCC5.891, respectively; B was purchased from the China Agricultural Microbiological Collection with accession number ACCC51638. Each of the three strains of Phellinus linteus was inoculated onto potato dextrose agar (PDA) plates and incubated in a 27°C incubator for 7 days. Fresh colonies were transferred to new PDA plates for purification and culture for 10 days. A 6 mm diameter bacterial cake was then obtained using a microporator for later use.

[0027] (2) Screening of bacterial strains:

[0028] 1. Preparation of broken rice flour: The broken rice produced in the rice production process is crushed by a grinder, sieved (35 mesh), and obtained broken rice flour, which is stored at room temperature for later use.

[0029] 2. Preparation of basal culture medium: Weigh 5 g of broken rice flour and 1 g of peptone into a 250 mL Erlenmeyer flask, add 100 mL of distilled water, sterilize at 121°C for 15 min, and cool for later use.

[0030] 3. Determination of Dry Weight of Broken Rice Fermented with Phellinus Igneus: Filter the fermentation broth using a constant-weight quantitative filter paper. Collect the filtrate in a 50mL centrifuge tube for determination of total flavonoids. Wash the filter residue thoroughly with ultrapure water and dry it at 60°C to a constant weight. Weigh and calculate the difference in mass before and after fermentation to determine the dry weight. The dry weight of the three strains of Phellinus Igneus after fermentation was significantly different: B > C > A.

[0031] 4. Determination of Total Flavonoids in Fermented Broth of Phellinus Iiformis: The total flavonoids (calculated as rutin) in the filtered fermentation broth were extracted with ethanol and then reacted with aluminum salts under weak alkaline conditions to form a chelate. The color was developed by adding sodium hydroxide solution, and the absorbance was measured at a wavelength of 510 nm. Within a certain concentration range, the absorbance of the chelate was directly proportional to the total flavonoid content, and quantification was performed using a standard curve method.

[0032] (3) Reserve reagent configuration:

[0033] Reagent 1, ethanol solution (7+3): Measure 70 mL of anhydrous ethanol and add it to 30 mL of purified water, mix well and set aside.

[0034] Reagent 2, sodium nitrite solution (50 g / L): Accurately weigh 5.00 g of sodium nitrite into a 100 mL volumetric flask, add appropriate amount of water to dissolve, add water to the scale, shake well, and set aside.

[0035] Reagent 3, aluminum nitrate solution (100 g / L): Accurately weigh 10.00 g of aluminum nitrate into a 100 mL volumetric flask, add appropriate amount of water to dissolve, add water to the scale, shake well, and set aside.

[0036] Reagent 4, sodium hydroxide solution (40 g / L): Accurately weigh 8.00 g of sodium hydroxide into a 200 mL volumetric flask, add appropriate amount of water to dissolve, dilute to the mark with water, shake well, and set aside.

[0037] Reagent 5, rutin standard (C27H30O16, CAS No.: 153-18-4): purity ≥ 98.0%, or a standard substance certified by the state and awarded a standard substance certificate.

[0038] (1) Sample extraction: Weigh approximately 5 mL of sample solution into a 50 mL centrifuge tube, add 40 mL of 70% ethanol solution, and sonicate for 40 minutes while constantly shaking the tube. After sonication, remove the tube, wipe dry the water stains on the outside, and dilute to 50 mL with 70% ethanol. After dilution, shake the sample solution and centrifuge it at 5000 rpm for 10 minutes. Remove the supernatant and test it.

[0039] (2) Sample determination: The total flavonoid content was determined by measuring the absorbance at a wavelength of 510 nm using an ultraviolet spectrophotometer. The sample solution was aspirated according to the optimal aspiration volume of the pre-experimental experiment and tested. At the same time, a rutin standard solution was prepared and loaded onto the instrument. A linear analytical equation was developed using the reference standard to calculate the total flavonoid content.

[0040] (3) Calculation formula: ω = [(ρ - ρ0) × V1 × V3] / (m × V2 × 1000)

[0041] ω——The value of the total flavonoids content in the sample, in milligrams per gram (mg / g)

[0042] ρ——The mass concentration of total flavonoids in the sample solution obtained from the standard curve, in milligrams per liter (mg / L)

[0043] ρ0——The mass concentration of total flavonoids in the blank test solution obtained from the standard curve, in milligrams per liter (mg / L)

[0044] V1——The volume of the extraction solution added to the sample and fixed to volume, in milliliters (mL)

[0045] V3——The final fixed volume of the sample, in milliliters (mL)

[0046] m——Numerical value of the mass of the sample, in grams (g)

[0047] V2 - the volume of the extract, in milliliters (mL)

[0048] During this test: V1=50, V2=10, V3=2, m=5g.

[0049] (4) Calculation results: After the three strains of Phellinus igniarius fermented broken rice, the total flavonoids content in the fermentation filtrate was different. Among them, the total flavonoids content in the filtrate produced by fermenting broken rice with C was the highest. Figure 1 Based on dry cell weight, strain C of the three strains was most suitable for fermenting broken rice. Its fermentation filtrate contained 0.1089 mg / mL of total flavonoids, and its dry cell weight was 1.27 g, surpassing the other strains. Therefore, strain C (Waning Phellinus 5.891) was selected as the fermentation strain.

[0050] Example 2

[0051] This example is about the screening of a high-yield flavonoid culture medium for fermenting broken rice with Phellinus igniarius: a single-factor test was used to screen the formula of the culture medium for fermenting broken rice with Phellinus igniarius.

[0052] (1) Carbon source: Weigh 5g of broken rice flour in a 250mL triangular flask, add 1g of peptone and 0.1g of magnesium sulfate, use glucose, maltose, sucrose and soluble starch as single factors, add carbon source at levels of 1, 1.25, 1.5, 1.75 and 2g / 100mL of distilled water, inoculate at 5 bacterial cakes / 100mL, and culture in a shake flask at 180r / min and 27°C for 10 days. Determine the dry weight of the bacteria after fermentation and the total flavonoids content of the fermentation filtrate according to the method in Example 1. It was determined that when maltose was used as the carbon source, the total flavonoids content in the fermentation filtrate was better than that of other carbon sources. When the maltose addition amount was 2.00g / 100mL, the total flavonoids content was the highest, reaching 2.70mg / mL. For details, see Figure 2 and Figure 3 .

[0053] (2) Nitrogen source: Weigh 5g of broken rice flour in a 250mL conical flask, add 1.5g of glucose and 0.1g of magnesium sulfate, use peptone, urea and ammonium sulfate as single factors, add nitrogen source at levels of 0.5, 0.75, 1, 1.25 and 1.5g / 100mL of distilled water, inoculate at 5 bacterial cakes / 100mL, and culture in a shake flask at 180r / min and 27℃ for 10 days. Determine the dry weight of the bacteria after fermentation and the total flavonoids content of the fermentation filtrate according to the method in Example 1. It was determined that when peptone was used as the nitrogen source, the total flavonoids content in the fermentation filtrate was better than that of other nitrogen sources. When the amount of peptone added was 1.00g / 100mL, the total flavonoids content was the highest, reaching 0.8036mg / mL. For details, see Figure 4 and Figure 5 .

[0054] (III) Inorganic salts: Weigh 5 g of broken rice flour into a 250 mL Erlenmeyer flask, add 1.5 g of glucose and 1 g of peptone, use magnesium sulfate, potassium dihydrogen phosphate, and ferrous sulfate as single factors, add inorganic salts at 0.06, 0.08, 0.1, 0.12, and 0.14 g / 100 mL of distilled water, inoculate at 5 bacterial cakes / 100 mL, and shake culture at 180 rpm and 27°C for 10 days. Determine the dry weight of the fermented cells and the total flavonoid content of the fermentation filtrate according to the method in Example 1. Figure 6 and Figure 7 The results showed that when inorganic salts were used as nitrogen sources, the total flavonoid content in the fermentation filtrate was superior to that of other nitrogen sources. When the addition amount of magnesium sulfate was 0.12 g / 100 mL, the total flavonoid content was the highest, reaching 0.9258 mg / mL.

[0055] Example 3

[0056] This example is the total flavonoids content of broken rice fermented with Phellinus igniarius: 5g of broken rice powder was weighed in a 250mL triangular flask, 2.0g of maltose, 1.0g of peptone and 0.12g of magnesium sulfate were added, 5 bacterial cakes were inoculated, and the culture was carried out at 180r / min and 27°C for 10 days. The dry weight of the bacteria after fermentation and the total flavonoids content of the fermentation filtrate were determined according to the method in Example 1. It was determined that the total flavonoids content was 1.1430mg / mL, which was higher than that of the basal medium fermentation; the dry weight of the bacteria was 1.7227g, which was higher than that of the basal medium fermentation 0.1089mg / mL. See Table 1 for details:

[0057] Table 1

[0058] Total flavonoids (mg / mL) 1.1430±0.04b Dry weight of bacteria (g) 1.7227±0.11a

[0059] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the present invention's specification and drawings, and direct or indirect application of the above technical solutions to other related technical fields, are all included in the scope of patent protection of the present invention.

Claims

1. A culture medium suitable for fermenting broken rice with Phellinus igniarius to produce high flavonoids, prepared by the following method: Weigh 5 g of broken rice flour into a 250 mL Erlenmeyer flask, add 2.0 g of maltose, 1.0 g of peptone and 0.12 g of magnesium sulfate, inoculate 5 bacterial cakes, and culture in a shake flask for 10 days to obtain the product.

2. The culture medium according to claim 1, wherein: The particle size of the broken rice flour is 35 meshes.

3. The culture medium according to claim 1, wherein: The mushroom cake is prepared by the following method: The Vaninian linterella was inoculated onto a potato dextrose agar plate and cultured in a 27°C incubator for 7 days. Fresh colonies were picked and transferred to a new potato dextrose agar plate for purification and culture for 10 days. A 6 mm diameter bacterial cake was taken with a puncher for later use. Vaninian linterella was purchased from the China Microorganism Collection Center with the collection number CGMCC5.

891.

4. The culture medium according to claim 1, wherein: The shaking flask culture speed is 180 r / min and the culture temperature is 27°C.

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