A method for extracting quercetin by using phomopsis XP-8

Abstract

The present application relates to the technical field of quercetin extraction, and particularly relates to a method for extracting quercetin by using phomopsis XP-8, which comprises the following steps: step one, a little phomopsis XP-8 bacteria in a test tube is picked up by using an inoculation needle, and is inoculated on a PDA plate; step two, five agar blocks with mycelium are punched from the PDA culture of the cultured bacteria by using a puncher with a diameter of 5 mm, and are transferred into 100 mL of PDB culture medium; step three, the bacteria obtained in step two are centrifuged and washed with sterile water for three times; glucose is used as a substrate, and quercetin solution is added for induction; step four, the reaction solution in step three is centrifuged, the bacteria are collected, washed with sterile water for three times, and quercetin solution is added for adsorption; and step five, the reaction solution in step four is centrifuged, the bacteria are collected, and are added into anhydrous ethanol for desorption. The phomopsis XP-8 used in the method is safe and cheap in source, and the operation method is simple.

Description

Technical Field

[0001] This invention relates to the technical field of quercetin extraction, specifically to a method for effectively extracting quercetin using Pseudomonas xylostellae XP-8. Background Technology

[0002] Quercetin is a flavonoid compound with various biological activities, including antioxidant, antiviral, antibacterial, anti-inflammatory, hepatoprotective, hypotensive, and anticancer effects. However, its solubility and bioavailability are very low. Classic extraction methods for quercetin, such as heating, boiling and reflux, steam distillation, or organic solvent impregnation, have been used for decades. These older methods are simple, robust, and require minimal equipment, but they are labor-intensive, time-consuming, and consume a lot of solvents, often using toxic organic solvents, which increases the need for space and equipment.

[0003] Furthermore, due to legislative requirements in the food and pharmaceutical industries, the use of harmful organic solvents is prohibited not only in products but also throughout the manufacturing process. This, coupled with increased awareness of environmental responsibility and green development, has led to a shift in quercetin extraction methods towards greener processes. Modern extraction methods include ultrasonic extraction, supercritical fluid extraction, near-critical or supercritical water extraction, and microwave-assisted extraction. These alternative technologies typically require less organic solvent, but they are technically demanding and costly. Summary of the Invention

[0004] To overcome the above-mentioned problems, this invention proposes a method for extracting quercetin using Pseudomonas xylostellae XP-8. This technology is not only simple to operate, has a short extraction time and high extraction efficiency, but also uses environmentally friendly solvent ethanol to elute quercetin from the mycelium, which is in line with the trend of economic, green and sustainable development.

[0005] The technical solution of the present invention is as follows:

[0006] A method for extracting quercetin using *Pseudomonas stolonifera* XP-8 includes the following steps:

[0007] Step 1: Use an inoculation needle to pick up a small amount of *Pseudomonas stolonifer* XP-8 cells preserved on the test tube slant and inoculate them onto a PDA plate;

[0008] Step 2: Using a 5mm diameter punch, take 5 agar blocks with mycelia from the cultured PDA culture and transfer them to 100mL of PDB medium.

[0009] Step 3: Collect the bacterial cells obtained in Step 2 by centrifugation, wash three times with sterile water, and induce induction with quercetin solution using glucose as a substrate;

[0010] Step 4: Centrifuge the reaction solution from Step 3, collect the bacterial cells, wash three times with sterile water, and add quercetin solution for adsorption;

[0011] Step 5: Centrifuge the reaction solution from Step 4, collect the bacterial cells, and add them to anhydrous ethanol for desorption.

[0012] Furthermore, the culture conditions in step one are static culture at 28°C for 5 days.

[0013] Furthermore, in step two, the culture conditions are 240 rpm and 28°C for 3 days.

[0014] Furthermore, in step three, the glucose concentration is 20 g / L, the quercetin concentration is 50 mg / L, the temperature is 28°C, the rotation speed is 220 r / min, and the reaction time is 24 h.

[0015] Furthermore, in step four, the centrifugation speed is 8000 rpm for 10 minutes.

[0016] Furthermore, in step five, the concentration of anhydrous ethanol is 100%.

[0017] The beneficial technical effects of the present invention are as follows:

[0018] The method of this invention utilizes *Phyllostachys xylophilus* XP-8, a safe and inexpensive fungus, and its operation is simple. This method can achieve adsorption equilibrium of quercetin within a short time (30 minutes). By adjusting conditions such as quercetin concentration, cell volume, total reaction volume, pH, temperature, and reaction time, 100% adsorption of quercetin can be achieved. The desorption reagent used in this invention is the environmentally friendly solvent ethanol. The desorption efficiency of *Phyllostachys xylophilus* XP-8 for quercetin reaches 91.53%. Attached Figure Description

[0019] Figure 1 The effect of mycelium dosage on quercetin adsorption efficiency.

[0020] Figure 2 The effect of initial quercetin concentration on adsorption efficiency.

[0021] Figure 3 The effect of reaction speed on adsorption efficiency.

[0022] Figure 4 The effect of pH of the reaction solution on the adsorption effect.

[0023] Figure 5 The effect of reaction time on adsorption efficiency.

[0024] Figure 6 The effect of reaction temperature on adsorption efficiency.

[0025] Figure 7 The effect of initial quercetin concentration on the desorption efficiency of quercetin.

[0026] Figure 8 The effect of bacterial dosage on the desorption effect of quercetin.

[0027] Figure 9 The effect of reaction speed on the desorption of quercetin.

[0028] Figure 10 The effect of pH of the reaction solution on the desorption and adsorption of quercetin.

[0029] Figure 11 The effect of reaction time on the desorption of quercetin.

[0030] Figure 12 The effect of reaction temperature on the desorption of quercetin.

[0031] Figure 13 The effect of bacterial cell freshness on the desorption efficiency of quercetin. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Example:

[0034] A method for extracting quercetin using *Pseudomonas stolonifera* XP-8 includes the following steps:

[0035] Step 1: Using an inoculation needle, pick up a small amount of *Pseudomonas aeruginosa* XP-8 cells preserved on a test tube slant and inoculate them onto a PDA plate. Incubate at 28°C for 5 days. Once the white mycelium has covered the entire PDA plate, remove it and store it in a 4°C refrigerator for later use.

[0036] Step 2: Using a 5mm diameter punch, take 5 agar blocks with mycelia from the cultured PDA culture and transfer them to 100mL of PDB medium. Place the inoculum in a temperature-controlled rotary shaker and culture for 3 days at 240rpm and 28℃. Take all the culture and separate the mycelia from the liquid culture by vacuum filtration. After washing three times with sterile water, collect the mycelia.

[0037] Step 3: On the 4th day, the mycelium collected in Step 2 was transferred to a 20 g / L glucose aqueous solution and a 50 mg / L quercetin solution was added. The mycelium was incubated at 28℃ and 240 r / min for 24 h. After the reaction was completed, the mycelium was centrifuged at 8000 rpm for 10 min, collected, and washed three times with sterile water for later use.

[0038] Adsorption conditions:

[0039] (1) The bacterial cell dosages were 10 mg, 15 mg, 20 mg, 50 mg, 80 mg, 100 mg, 150 mg, and 200 mg, respectively. Other reaction conditions were set as follows: initial quercetin concentration of 100 mg / L, solution pH of 6.0, and reaction volume of 0.005 L. The mixed reaction solution was placed at 28 °C and 240 rpm for 3 h.

[0040] (2) The initial concentrations of quercetin in the reaction solution were 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, 150 mg / L, 180 mg / L, and 200 mg / L, respectively. Other reaction conditions were set as follows: the amount of wet mycelium of *Pseudomonas aeruginosa* XP-8 was 10 g / L, the pH of the solution was 6.0, and the volume of the reaction solution was 0.005 L. The mixed reaction solution was placed at 28℃ and 240 rpm for 3 h.

[0041] (3) The reaction speeds were 0 rpm, 180 rpm, and 240 rpm, while other reaction conditions were maintained as follows: initial quercetin concentration of 100 mg / L, amount of wet mycelium of *Pseudomonas aeruginosa* XP-8 of 10 g / L, solution pH of 6.0, and reaction volume of 0.005 L. The reaction solution was placed at 28℃ and 240 rpm for 3 h.

[0042] (4) The pH of the reaction solution was adjusted to 5.0, 6.0, 7.0 and 8.0 respectively using phosphoric acid and NaOH solution. The initial concentration of quercetin was 100 mg / L, the amount of mycelium used was 10 g / L, and the reaction volume was 0.005 L. The reaction solution was placed at 28℃ and 240 rpm for 3 h.

[0043] (5) The initial concentration of quercetin in the reaction solution was 100 mg / L, the amount of wet mycelium of *Pseudomonas aeruginosa* XP-8 was 10 g / L, the reaction solution volume was 0.005 L, the pH was 6.0, and the reaction was carried out at 28℃ and 240 rpm. 1 mL of the centrifuged reaction solution was collected at each of the following time points (0 min, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, and 60 min). The residual quercetin concentration in the reaction solution was detected by high-performance liquid chromatography after filtration through a 0.22 μm filter membrane.

[0044] (6) The initial concentration of quercetin was 100 mg / L, the amount of wet mycelium of *Pseudomonas aeruginosa* XP-8 was 10 g / L, the total volume of the reaction solution was 0.005 L, and the pH value was 6.0. The reaction was carried out at 15℃, 18℃, 20℃, 25℃, 28℃, 30℃, and 35℃ with shaking at 240 rpm for 3 h. After the reaction was completed, 1 mL of the centrifuged reaction solution was collected and filtered through a 0.22 μm filter membrane. Finally, the residual quercetin concentration in each reaction system was measured by high performance liquid chromatography.

[0045] Desorption conditions:

[0046] (1) The wet mycelium that had been saturated with quercetin and prepared with initial quercetin concentrations of 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, 150 mg / L, 180 mg / L and 200 mg / L was desorbed with 100% anhydrous ethanol and placed at 28°C for 3 h. The pH of the solution was 6.0 and the total volume of the reaction solution was 0.005 L.

[0047] (2) 10 mg, 15 mg, 20 mg, 50 mg, 80 mg, 100 mg, 150 mg and 200 mg of wet mycelium that had reached quercetin saturation adsorption were desorbed with 100% anhydrous ethanol and placed at 28°C for 3 h. The pH of the solution was 6.0 and the total volume of the reaction solution was 0.005 L.

[0048] (3) The wet mycelium that has reached saturation adsorption of quercetin was desorbed using 100% anhydrous ethanol. The rotation speed was adjusted to 0 rpm, 180 rpm and 240 rpm respectively. The pH value of the solution was 6.0 and the total volume of the reaction solution was 0.005 L. The reaction was carried out at 28℃ for 3 h.

[0049] (4) The wet mycelium that has reached saturation adsorption of quercetin was desorbed using 100% anhydrous ethanol. The pH of the reaction solution was adjusted to 5.0, 6.0, 7.0 and 8.0 respectively using phosphoric acid and NaOH solution. The reaction solution was placed at 28℃ and allowed to stand for 3 hours. The total volume of the reaction solution was 0.005L.

[0050] (5) Select wet mycelia that have reached saturation adsorption of quercetin, and desorb them using 100% anhydrous ethanol. The reaction liquid volume is 0.005 L, pH is 6.0, and the reaction is allowed to stand at 28℃. Collect 1 mL of the centrifuged reaction liquid at each of the following time points (0 min, 2 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, and 60 min). Filter the solution through a 0.22 μm organic filter membrane and determine the quercetin concentration in the reaction solution using high performance liquid chromatography.

[0051] (6) Select wet mycelia that have reached saturation adsorption of quercetin, use 100% anhydrous ethanol as the desorption reagent, the reaction liquid volume is 0.005 L, the mass of wet mycelia of *Pseudomonas stolonifera* is 0.05 g, and the pH value is 6.0. The reaction was allowed to stand for 1 h at 15℃, 18℃, 20℃, 25℃, 28℃, 30℃, and 35℃. After the reaction, 1 mL of the centrifuged reaction liquid was collected and filtered through a 0.22 μm organic filter membrane. The concentration of quercetin in each reaction system was measured using high performance liquid chromatography (HPLC).

[0052] (7) The effects of quercetin on the mycelium of Pseudomonas xylostellae XP-8 stored at 4℃ for 7d, 20d, 30d, 50d and 60d respectively were evaluated under the following conditions: optimal adsorption (initial concentration of quercetin was 100mg / L, amount of Pseudomonas xylostellae XP-8 mycelium used was 10g / L, pH value was 6.0, reaction volume was 0.005L, and the reaction was allowed to stand at 28℃ for 30min) and desorption (100% anhydrous ethanol was used as the desorption reagent, and the wet mycelium after the reaction under the optimal adsorption conditions was placed at 28℃ and pH value was 5.0 for 35min for desorption, and the total reaction volume was 0.005L).

[0053] The adsorption effect of the above-mentioned *Pseudomonas stolonifera* XP-8 on quercetin was evaluated, and the results are as follows:

[0054] A. The amount of bacteria used affects the adsorption effect.

[0055] like Figure 1 As shown, the adsorption rate of quercetin gradually increases with the increase of the amount of wet mycelium used. When the amount of wet mycelium used is greater than 50 mg, the adsorption rate of quercetin reaches 100% complete adsorption.

[0056] B. The initial concentration of quercetin affects the adsorption effect.

[0057] like Figure 2 As shown, when the initial concentration of quercetin is low (0.03-0.33 mmol / L), the adsorption rate of quercetin by 10 g / L of wet mycelium of *Pseudomonas stolonifera* can reach 100%. When the concentration of quercetin increases from 0.33 mmol / L to 0.66 mmol / L, the adsorption rate of quercetin decreases to 85.01%. This indicates that the adsorption capacity of a certain amount of mycelium is limited, and a saturation equilibrium exists.

[0058] C. Rotation speed does not affect the adsorption effect.

[0059] like Figure 3As shown, the adsorption efficiency of quercetin by wet mycelium of Pseudomonas stolonifera XP-8 is independent of rotation speed, and 100% complete adsorption can be achieved under three rotation speed conditions: 0 rpm, 180 rpm and 240 rpm.

[0060] D. The mycelium has excellent natural pH adsorption effect.

[0061] like Figure 4 As shown, the adsorption of quercetin by the wet mycelium increased from pH 5.0 to 6.0, but the adsorption rate of quercetin remained unchanged with further increases in pH. Since the reaction solution itself has a pH of 6.0, pH adjustment is not required during the experiment, reducing experimental costs.

[0062] E. The adsorption process requires a certain amount of time.

[0063] like Figure 5 As shown, when the quercetin concentration is 100 mg / L, biosorption occurs rapidly within the first 10 minutes and reaches equilibrium at 30 minutes. If the reaction time is extended further, the quercetin adsorption rate remains basically unchanged, indicating that the optimal adsorption time for quercetin by wet mycelium of Pseudomonas xylostellae XP-8 is 30 minutes.

[0064] F. Excessively high temperatures are detrimental to quercetin adsorption.

[0065] Depend on Figure 6 It can be seen that the adsorption efficiency of quercetin increases significantly with the continuous rise in temperature, reaching a maximum of 98.33% at 28℃. With further increases in temperature, the adsorption rate of quercetin gradually decreases, even dropping to 7.83%. This indicates that the optimal temperature for the adsorption of quercetin by the wet mycelium of Pseudomonas xylostella XP-8 is 28℃, and further increases in temperature have an adverse effect on the adsorption efficiency of quercetin.

[0066] G. The initial concentration of quercetin affects the adsorption effect.

[0067] like Figure 7 As shown, when the initial concentration of quercetin increased from 0.03 mmol / L to 0.33 mmol / L, the desorption rate of quercetin gradually increased, reaching 91.53%. Further increasing the quercetin concentration significantly reduced the desorption efficiency. This indicates that when anhydrous ethanol is used as the desorbent, the saturation concentration for desorption of quercetin by *Phyllostachys spp.* mycelium is 0.33 mmol / L.

[0068] The desorption effect under different conditions was evaluated, and the results are as follows:

[0069] A. The amount of bacteria in the desorption solution affects the effect.

[0070] By fixing the amount of desorption solution and changing the amount of bacterial cells, the following results can be obtained: Figure 8As shown, when the amount of mycelium used increased from 10 mg to 50 mg, the desorption rate of quercetin gradually increased, reaching 91.53%. Further increasing the amount of mycelium used resulted in a significant decrease in the desorption rate of quercetin. Therefore, the optimal amount of mycelium used for the desorption of quercetin by *Pseudomonas xylostella* XP-8 wet mycelium is 50 mg.

[0071] B. Reaction speed affects desorption efficiency

[0072] like Figure 9 As shown, the desorption rate of quercetin gradually decreased with increasing rotation speed. This indicates that the optimal rotation speed for the desorption of quercetin by *Pseudomonas aeruginosa* XP-8 wet mycelium is 0 rpm.

[0073] C. pH affects desorption efficiency

[0074] like Figure 10 As shown, the desorption rate of quercetin decreased significantly with increasing pH value. When the pH value was 5, the wet mycelium achieved complete desorption of quercetin.

[0075] D. Reaction time affects desorption efficiency

[0076] like Figure 11 As shown, when the quercetin concentration was 100 mg / L, the desorption of quercetin by the wet mycelium occurred within the first 15 minutes, reaching equilibrium at 35 minutes. Further extending the reaction time did not significantly increase the desorption rate of quercetin, indicating that the optimal desorption time for quercetin by the wet mycelium of *Pseudomonas thunbergii* was 35 minutes.

[0077] E. Reaction temperature affects desorption.

[0078] Depend on Figure 12 It can be seen that as the temperature rises, the desorption rate of quercetin gradually increases, reaching a maximum of 90.60% at 28℃. With further increases in temperature, the desorption rate of quercetin decreases significantly, indicating that the optimal temperature for the desorption of quercetin by wet mycelium of Pseudomonas stolonifera is 28℃.

[0079] F. The freshness of the mycelium affects the adsorption and desorption effects.

[0080] Depend on Figure 13 It was found that the wet mycelium of *Pseudomonas stolonifera* XP-8, stored at 4℃ for up to two months, maintained its adsorption capacity for quercetin, achieving 100% complete adsorption. However, with prolonged storage, the desorption capacity of the wet mycelium for quercetin significantly decreased. The desorption rate of quercetin from fresh wet mycelium stored for up to one week was 97.73%, but after one month, the desorption rate was only half, and after two months, it was only one-third. This indicates that storing *Pseudomonas stolonifera* wet mycelium at 4℃ for no more than 7 days is the optimal experimental time for its adsorption and desorption capacity for quercetin.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. It should be noted that any improvements and modifications made by those skilled in the art without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for extracting quercetin using *Pseudomonas stolonifera* XP-8, characterized in that, Includes the following steps: Step 1: Use an inoculation needle to pick up a small amount of *Pseudomonas stolonifer* XP-8 cells preserved on the test tube slant and inoculate them onto a PDA plate; Step 2: Using a 5 mm diameter punch, take 5 agar blocks with hyphae from the cultured PDA culture and transfer them to 100 mL of PDB medium. Step 3: Collect the bacterial cells obtained in Step 2 by centrifugation, wash three times with sterile water, and induce induction with quercetin solution using glucose as a substrate; Step 4: Centrifuge the reaction solution from Step 3, collect the bacterial cells, wash three times with sterile water, and add quercetin solution for adsorption; Step 5: Centrifuge the reaction solution from Step 4, collect the bacterial cells, and add them to anhydrous ethanol for desorption. The adsorption conditions in step four include: a wet mycelium usage of more than 10 g / L, an initial quercetin concentration of 0.03-0.66 mmol / L, a natural pH of 5.0-6.0 for the mycelium, an adsorption time of 30 min, and an adsorption temperature of 28℃. The desorption conditions in step five include: a bacterial cell dosage of 10 g / L, a pH of 5.0, a desorption time of 35 min, a desorption temperature of 28 ℃, and the freshness of the mycelium used being kept at 4 ℃ for no more than 7 days. In step three, the glucose concentration is 20 g / L, the quercetin concentration is 50 mg / L, the temperature is 28 ℃, the rotation speed is 220 r / min, and the reaction time is 24 h.

2. The method for extracting quercetin using *Pseudomonas stolonifera* XP-8 according to claim 1, characterized in that: The culture conditions in step one are static culture at 28℃ for 5 days.

3. The method for extracting quercetin using *Pseudomonas stolonifera* XP-8 according to claim 2, characterized in that: In step two, the culture conditions are 240 rpm and 28°C for 3 days.

4. The method for extracting quercetin using *Pseudomonas aeruginosa* XP-8 according to claim 3, characterized in that: In step four, the centrifugation speed is 8000 rpm for 10 minutes.

5. The method for extracting quercetin using *Pseudomonas stolonifera* XP-8 according to claim 4, characterized in that: In step five, the concentration of anhydrous ethanol is 100%.

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