Method for reinforcing luteolin and resveratrol by fermenting peanut shell with fungi and product and application thereof

Abstract

This invention belongs to the field of functional food fermentation technology, specifically relating to a method for fortifying peanut shells with luteolin and resveratrol through fungal fermentation, the resulting products, and their applications. The specific steps of this method are as follows: First, it was found that the relative contents of luteolin and resveratrol increased after co-fermentation of Ganoderma lucidum and peanut shells. Subsequently, by optimizing the fermentation and extraction conditions, the contents of resveratrol and luteolin in the fermented extract were increased. This method utilizes the bio-fermentation of edible and medicinal fungi, which utilizes components such as lignin in peanut shells to promote the growth of Ganoderma lucidum, and also releases luteolin and resveratrol, which are difficult to extract from peanut shells. The fermented extract contains both beneficial substances from Ganoderma lucidum and the two beneficial substances, luteolin and resveratrol, from peanut shells, with a 28.57% increase in resveratrol content and a 43.08% increase in luteolin content.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for fortifying peanut shells with luteolin and resveratrol through fungal fermentation, the resulting products, and their applications. Technical Background Peanuts, as a major oilseed crop, produce tens of millions of tons of peanut shells annually as a processing byproduct. Currently, peanut shells are mostly discarded, burned, or used as cheap animal feed, resulting in resource waste and environmental pollution. In fact, peanut shells are rich in cellulose, hemicellulose, lignin, and small amounts of natural polyphenols, making them a potential low-cost natural raw material with the potential for high-value development.

[0002] Luteolin and resveratrol are both natural polyphenolic compounds with significant biological activity. Luteolin possesses antioxidant, anti-inflammatory, antibacterial, and immunomodulatory effects, and has broad application prospects in the food and pharmaceutical fields. Resveratrol, on the other hand, has antioxidant, anti-aging, and cardiovascular protective effects, and is an important active ingredient in functional foods. However, the content of luteolin and resveratrol in natural peanut shells is extremely low, making direct extraction difficult to meet the needs of industrial production.

[0003] Existing methods for enhancing plant-derived polyphenols mainly include chemical synthesis, direct extraction, and biotransformation. Chemical synthesis suffers from harsh reaction conditions, low product purity, and the potential for toxic byproducts, limiting its application in the food industry. Direct extraction relies on large amounts of organic solvents, resulting in low extraction efficiency, high costs, and potential secondary environmental pollution. Edible and medicinal fungal fermentation, as a green biotransformation technology, can degrade macromolecules in peanut shells through enzymes produced by fungal metabolism, while simultaneously synthesizing or transforming target active ingredients. It offers advantages such as mild conditions, environmental friendliness, and high product safety.

[0004] Current research includes studies on the fermentation of peanut shells by fungi such as Aspergillus niger to prepare dietary fiber. However, no methods have been reported for fortifying peanut shells with luteolin and resveratrol through fermentation by edible and medicinal fungi to prepare polyphenolic functional foods. Therefore, developing an efficient and green fermentation process for edible and medicinal fungi to achieve high-value utilization of peanut shell resources and targeted fortification of active polyphenolic components has significant practical and market value. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for fortifying peanut shells with luteolin and resveratrol through fungal fermentation.

[0006] The present invention also provides a Ganoderma lucidum fermented peanut shell extract prepared by the above method.

[0007] Another object of the present invention is to provide the application of the above-mentioned Ganoderma lucidum fermented peanut shell extract.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides a method for fortifying peanut shells with luteolin and resveratrol through fungal fermentation, comprising the following steps: (1) Grind the peanut shells into granules and sieve them to obtain peanut shell powder; (2) Add glucose, yeast powder and purified water to peanut shell powder while stirring. Stir well and then sterilize to obtain peanut shell culture medium. (3) Inoculate the bottom of the peanut shell culture medium with Ganoderma lucidum, stir evenly, seal and ferment to obtain the fermented product of Ganoderma lucidum fermented with peanut shell; (4) Soak the dried fermented material in ethanol solution, filter and centrifuge to obtain supernatant, rotary evaporate the obtained supernatant, freeze dry after rotary evaporation to obtain Ganoderma lucidum fermented peanut shell extract.

[0009] Preferably, in step (1), the peanut shell powder is obtained by passing it through a 50-mesh sieve.

[0010] Preferably, in step (2), the proportion of purified water is 70%, the amount of glucose added accounts for 2-3% of the total weight of the peanut shell culture medium, and the amount of yeast powder added accounts for 1-2% of the total weight of the peanut shell culture medium.

[0011] Preferably, in step (2), the sterilization is performed by sealing and autoclaving at 121-125°C for 30-35 minutes.

[0012] Preferably, in step (3), the amount of Ganoderma lucidum inoculated into the peanut shell culture medium is 13-15%.

[0013] Preferably, in step (3), the fermentation culture is carried out in an incubator at 26-28℃ for 14-18 days.

[0014] Preferably, in step (4), the volume ratio of the fermented product to the ethanol solution is 1:20-25; and the concentration of the ethanol solution is 90-100% ethanol (V / V).

[0015] Preferably, in step (4), the soaking is performed under ultrasonic conditions for 30-40 minutes.

[0016] The present invention also provides a fermented peanut shell product of Ganoderma lucidum prepared by the above method.

[0017] Another objective of this invention is to provide the application of the above-mentioned Ganoderma lucidum fermented peanut shell ferment in the preparation of polyphenolic functional foods.

[0018] This invention utilizes specific fermentation and extraction conditions to increase the content of resveratrol and luteolin in the fermented extract. This method employs the bio-fermentation of edible and medicinal fungi, utilizing lignin and other components in peanut shells to promote the growth of Ganoderma lucidum, while simultaneously releasing luteolin and resveratrol, which are difficult to extract from peanut shells. The resulting fermented extract contains both beneficial substances from Ganoderma lucidum and the two beneficial substances from peanut shells: luteolin and resveratrol. Furthermore, the resveratrol content of the extract is increased by 28.57%, and the luteolin content by 43.08%.

[0019] The Ganoderma strain used in this invention is commercially available Ganoderma lucidum from Taishan. Its appearance characteristics are as follows: in the early stage, the hyphae are white or light yellow, and the tips of the hyphae are branched in a dendritic manner; in the middle stage, colonies are formed on the culture medium, and the older hyphae in the center of the colony secrete pigment, making the hyphae yellow; when the hyphae age, the colony changes from yellow to dark yellow.

[0020] The beneficial effects of this invention are as follows: (1) This invention is the first to use the edible and medicinal fungus Ganoderma lucidum to ferment peanut shells, which can reduce the content of substances that are not easily absorbed in peanut shells, thus making peanut shells more usable. (2) This invention provides a method for preparing extracts of luteolin and resveratrol by fermenting peanut shells with edible and medicinal fungi for the first time; the synergistic enzymatic hydrolysis can directionally increase the content of luteolin and resveratrol. Compared with unfermented peanut shells, the content of the two components is increased, which is far superior to the traditional extraction method. Attached Figure Description

[0021] Figure 1 HPLC chromatograms and standard curves for resveratrol and luteolin standards; Figure 2 The results are from single-factor experiments on resveratrol and luteolin. Figure 3 Optimization results for resveratrol and luteolin PB experiments; Figures 4-7 The results of response surface methodology optimization for resveratrol and luteolin are presented. Specific implementation methods To make the objectives, technical means, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of this invention is not limited to the embodiments described.

[0022] Example 1: Preparation of Ganoderma lucidum fermented peanut shell extract 1. Preparation of Ganoderma lucidum seed liquid Prepare PDY seed culture medium (200 g / L potato, 20 g / L glucose), dispense into Erlenmeyer flasks, autoclave at 121℃ for 20 min, and cool to room temperature. Use an inoculation needle to pick 1-2 loops of Ganoderma lucidum mycelium from the activated culture medium and inoculate it into the seed culture medium. Place the flasks in a shaker at 25-28℃ and 150-180 r / min for 5-7 days to obtain a seed solution with uniform growth.

[0023] 2. Fermentation of peanut shells (1) Crushing peanut shells: Crush the cleaned peanut shells and sieve them through a 50-mesh sieve to obtain peanut shell powder; (2) Add a certain amount of peanut shell powder to the prepared sterile water (mass ratio 10:21, total mass 70%), glucose (total mass 2-3%), and yeast powder (total mass 1-2%). Stir while adding the powder until the peanut shell powder is neither loose nor clumped, but rather tightly clumped together and dispersed when released. (3) Put the prepared peanut shells into a box, add plastic sealing film, cover and seal, and put it into an autoclave for high-pressure steam sterilization at 121 °C for 30 min; (4) Fermentation of peanut shells: Inoculate the Ganoderma lucidum seed liquid into the peanut shell culture medium (inoculation amount 15%), inoculate it into the bottom of the peanut shell as much as possible, stir evenly, cover with plastic sealing film, and seal; culture in an incubator at 27 ℃ for 16 days, and then dry to constant weight to obtain dried fermented product.

[0024] 3. Extraction The dried fermented material was ultrasonicated with 22 times its volume of 95% ethanol (V / V) for 35 min and then centrifuged to obtain the supernatant. The supernatant was then rotary evaporated and freeze-dried using a freeze dryer. After grinding, a powdered, easily preserved Ganoderma lucidum fermented peanut shell extract was obtained.

[0025] Example 2: Determination of the content of luteolin and resveratrol in Ganoderma lucidum fermented peanut shell extract The contents of luteolin and resveratrol in the fermented peanut shell extract of Ganoderma lucidum prepared in the examples were determined by high performance liquid chromatography (HPLC).

[0026] Experimental results are as follows Figure 1 As shown, standard curves for resveratrol and luteolin content were obtained based on HPLC chromatographic results. The resveratrol standard curve was y = 55040x - 26.783, R² = 0.9994; the luteolin standard curve was y = 1590.5x + 19.036, R² = 0.9993, which were used for subsequent detection of resveratrol and luteolin content in fermentation products.

[0027] Example 3: Single-factor experimental results First, establish the standard single-factor conditions for Ganoderma lucidum fermentation: inoculum size (2-15%), fermentation time (5-25 days), fermentation temperature (21-31℃), moisture content (30-90%), carbon source, and nitrogen source. Compare these with unfermented peanut shells to determine suitable carbon and nitrogen sources as glucose and yeast powder, respectively. Then, determine the ratio between the two. Subsequently, based on the initial extraction method, establish the standard single-factor experiments for extraction: extraction reagent (common reagents for extracting resveratrol and luteolin), extraction time (ultrasonic 10-50 min), and material-to-liquid ratio (1:5-1:25).

[0028] Experimental results are as follows Figure 2 As shown, the selected fermentation time was 14-18 days, the inoculum size was 13-15%, the temperature was 26-28 ℃, the water content was 60-70%, the glucose content was 2-3%, the yeast powder (oxoid, Thermo Fisher) content was 1-2%, the ethanol content was 90-100%, the extraction time was 30-40 min, and the material-to-liquid ratio was 1:20-1:25 for subsequent experiments.

[0029] Example 4: PB Experiment The PB experiment was developed based on the results of the single-factor experiment in Example 3. The PB experiment design and response values ​​are shown in Table 1.

[0030] Table 1. PB Experimental Design and Response Values The results of the PB experiment are shown in Tables 2 and 3. Figure 3 As shown, the F-value of the fermentation condition model for resveratrol content is 17.11, and the R² is 0.9536; the F-value of the fermentation condition model for luteolin content is 25.55, and the R² is 0.9684, indicating that the model has high reliability. Furthermore, according to... Figure 3 Parthenocarpic analysis revealed that glucose content, yeast content, and fermentation time had a significant impact on resveratrol and luteolin levels after fermentation. Therefore, these three fermentation conditions were selected for subsequent response surface methodology optimization experiments.

[0031] Table 2. Analysis of variance of resveratrol content fermentation conditions in PB experiment. Table 3. Analysis of variance of fermentation conditions for luteolin content in PB experiment. Example 5: Response Surface Optimization Experiment Based on the single-factor experimental results in Example 3 and the PB experimental results in Example 4, a response surface optimization experiment was developed. The response surface optimization experimental design and response values ​​are shown in Tables 4 and 5.

[0032] Fermentation conditions were determined using a Box-Benhnken experimental design, with A (fermentation time), B (glucose content), and C (yeast content) as variables, and resveratrol and luteolin content as response values. The results are shown in Table 4. The results were input into DesignExpert 13 software for analysis and fitting, yielding quadratic regression equations for resveratrol content (Y1) and luteolin content (Y2), respectively: Y1 = 0.0533 + 3.62E -06 A-6.46E -07 B-2.58E -06 C-6.20E -06 AB-3.70E -07 AC+6.35E -06 BC-0.0001A 2 -0.0001B 2+ C 2 Y2=0.4515+0.0018A+0.003B+0.0003C+0.0001AB+AC+BC-0.0047A 2 -0.0073B 2 -0.001C 2 The extraction conditions were determined using a Box-Benhnken experimental design, with A (extraction time), B (ethanol concentration), and C (solid-liquid ratio) as variables, and resveratrol and luteolin content as response values. The results are shown in Table 5. The results were input into DesignExpert 13 software for analysis and fitting, yielding the quadratic regression equations for resveratrol content (Y1) and luteolin content (Y2), respectively: Y1 = 0.053 + 3.6138e -06 A-2.14e -06 B-4.08e -06 C-6.19e -06 AB-3.6e -07 AC4.34e -06 BC-5.35e -05 A²-7.63e -05 B²-1.147e -05 C² Y2=0.46+0.0018A+0.003B+0.0003C+9.19e -05 AB+AC-1.54e -05 BC -0.0046A²-0.0072B²-0.00093C² Table 4. Experimental design and response values ​​for fermentation condition response surface optimization. Table 5. Experimental Design and Response Values ​​for Extracting Conditional Response Surfaces The results of the content variance analysis of the fermentation conditional response surface model are shown in Tables 6 and 7. The p-value of the resveratrol content optimization model is <0.0001, and the p-value of the lack-of-fit term is 0.5007 > 0.05, indicating a good model fit. The R-value of the model... 2 =0.9881,R 2 Adj =0.9728, indicating a good correlation between the theoretical and actual measured values. The P-value of the luteolin content optimization model is <0.0001, and the P-value of the lack-of-fit term is 0.3412 >0.05, indicating a good model fit. The R-value of the model is... 2 =0.9989,R 2 Adj =0.9974, indicating a good correlation between the theoretical value and the actual measured value.

[0033] Table 6. Analysis of Variance in the Response Surface Optimization Experiment for Resveratrol Content Fermentation Conditions Table 7. Analysis of Variance in the Response Surface Optimization Experiment for Lactocerein Content Fermentation Conditions The results of the content variance analysis extracted from the conditional response surface methodology are shown in Tables 8 and 9. The p-value of the resveratrol content optimization model is <0.0001, and the p-value of the lack-of-fit term is 0.67 > 0.05, indicating a good model fit. The R-value of the model... 2 =0.9901,R 2 Adj =0.9775, indicating a good correlation between the theoretical and actual measured values. The P-value of the luteolin content optimization model is <0.0001, and the P-value of the lack-of-fit term is 0.3056 > 0.05, indicating a good model fit. The R-value of the model is... 2 =0.9988,R 2 Adj =0.9972, indicating a good correlation between the theoretical value and the actual measured value.

[0034] Table 8. Analysis of Variance in the Response Surface Optimization Experiment for Resveratrol Content Extraction Conditions Table 9. Analysis of Variance in the Response Surface Optimization Experiment for Lactolin Content Extraction Conditions Response surface methodology provides a clear visual representation of the impact of experimental factors and their interactions on the response values. 3D response surface plots and contour maps visually reflect the strength of these interactions, while the slope of the surface directly reflects the degree of influence of each factor on the response value; a steeper 3D plot indicates a more significant interaction between factors.

[0035] Response surface 3D plot and contour plot are generated by Figure 4 As shown, based on the response surface methodology for resveratrol and luteolin content, the interaction between fermentation time and glucose has a significant impact on the resveratrol and luteolin content. Figure 4 , Figure 5 The interaction between extraction time and ethanol concentration, as well as the solid-liquid ratio, had a significant impact on the content of resveratrol and luteolin. Figure 6 and Figure 7 ).

[0036] The fermentation and extraction conditions were optimized using DesignExpert 13 software. The results showed that the optimal model for resveratrol and luteolin content was: After optimization based on these conditions and the results of single-factor experiments, the fermentation conditions were as follows: inoculum size 12-15%, glucose content 2.52%, fermentation time 16 days, yeast content 1.48%, moisture content 65-70%, and fermentation temperature 27 ℃. The extraction conditions were: extraction time 35 min, ethanol concentration 95%, and material-to-liquid ratio 1:22. The contents were measured and significantly compared with those of unfermented peanut shells. It was found that the optimization significantly increased the contents of resveratrol and luteolin. The resveratrol content before fermentation was 0.042 mg / g, and after fermentation it was 0.054 mg / g; the luteolin content before fermentation was 0.325 mg / g, and after fermentation it was 0.465 mg / g, which were 28.57% and 43.08% higher than the control, respectively.

[0037] Based on previous experimental results, this invention uses the contents of resveratrol and luteolin as indicators. Single-factor experiments were conducted to screen fermentation and extraction processes, exploring the effects of fermentation and extraction conditions on the contents of resveratrol and luteolin in peanut shells. Based on the results of the single-factor experiments, PB experiments and response surface methodology were performed to investigate the influence of each factor and their interactions on the contents, establishing models for the contents of resveratrol and luteolin.

[0038] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for fortifying peanut shells with luteolin and resveratrol through fungal fermentation, characterized in that, Includes the following steps: (1) Grind the peanut shells into granules and sieve them to obtain peanut shell powder; (2) Add glucose, yeast powder and purified water to peanut shell powder while stirring. Stir well and then sterilize to obtain peanut shell culture medium. (3) Inoculate the bottom of the peanut shell culture medium with Ganoderma lucidum, stir evenly, seal and ferment to obtain the fermented product of Ganoderma lucidum fermented with peanut shell; (4) Soak the dried fermented material in ethanol solution, filter and centrifuge to obtain supernatant, rotary evaporate the obtained supernatant, freeze dry after rotary evaporation to obtain Ganoderma lucidum fermented peanut shell extract.

2. The method according to claim 1, characterized in that, In step (1), the peanut shell powder is obtained by passing it through a 50-mesh sieve.

3. The method according to claim 1 or 2, characterized in that, In step (2), the proportion of purified water is 70%, the amount of glucose added accounts for 2-3% of the total weight of the peanut shell culture medium, and the amount of yeast powder added accounts for 1-2% of the total weight of the peanut shell culture medium.

4. The preparation method according to claim 3, characterized in that, In step (2), the sterilization is performed by sealing and autoclaving at 121-125℃ for 30-35 minutes.

5. The preparation method according to claim 1, characterized in that, In step (3), the amount of Ganoderma lucidum inoculated into the peanut shell culture medium is 13-15%.

6. The method according to claim 1 or 5, characterized in that, In step (3), the fermentation culture is carried out in an incubator at 26-28℃ for 14-18 days.

7. The method according to any one of claims 1-6, characterized in that, In step (4), the volume ratio of the fermented product to the ethanol solution is 1:20-25; the concentration of the ethanol solution is 90-100% ethanol (V / V).

8. The method according to claim 7, characterized in that, In step (4), the soaking is performed under ultrasonic conditions for 30-40 minutes.

9. A fermented peanut shell product of Ganoderma lucidum prepared by the method according to any one of claims 1-8.

10. The application of the Ganoderma lucidum fermented peanut shell ferment as described in claim 9 in the preparation of polyphenolic functional foods.

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