A deep eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible mushroom polysaccharides and application thereof

Abstract

The application discloses a kind of eutectic solvent for simultaneously extracting water-soluble and non-water-soluble edible mushroom polysaccharides and its application, and belongs to the technical field of natural products extraction.The eutectic solvent for simultaneously extracting water-soluble and non-water-soluble edible mushroom polysaccharides according to the application is composed of n-octanoic acid and sodium octanoate;The molar ratio of n-octanoic acid and sodium octanoate is 2-3:1.The method of the application can realize the hydrophilic-hydrophobic switching of eutectic solvent by adjusting the pH value of the extraction system, and simultaneously extract water-soluble and non-water-soluble polysaccharides;The yield of extracted polysaccharides is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and in particular to a low eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible fungus polysaccharides and applications thereof. Background Art

[0002] Golden Ear Naematelia aurantialba, Tremella serrata (NA) belongs to the Tremellales order, Ototrichum family, Ototrichum genus, and is parasitic on the fungus Tremella serrata. Its fruiting body is golden in appearance, resembling a human brain and possessing a golden hue. As a valuable edible and medicinal fungus, it is rich in nutrients, including polysaccharides, fats, minerals, and carotenoids. Polysaccharides are one of its most important bioactive components, exhibiting excellent antioxidant, hypoglycemic, hypolipidemic, and anti-inflammatory properties.

[0003] Common extraction solvents for natural polysaccharides include water, acids, and alkalis. These traditional solvents generally produce low yields of polysaccharides. In recent years, deep eutectic solvents (DES) have been widely used as a new green solvent. These solvents are composed of a specific mixture of hydrogen bond acceptors and hydrogen bond donors, and their properties are similar to those of ionic liquids. Currently, there are reports on the application of DES in polysaccharide extraction, but these methods only replace traditional solvents for the extraction of water-soluble polysaccharides, without fully utilizing the hydrophilic and hydrophobic properties of DES to effectively extract and separate hydrophilic and hydrophobic active substances. Summary of the Invention

[0004] The purpose of the present invention is to provide a deep eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible fungus polysaccharides and its application. The deep eutectic solvent of the present invention can simultaneously and efficiently extract water-soluble and water-insoluble polysaccharides.

[0005] The invention first provides a low eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible fungus polysaccharides. The low eutectic solvent consists of n-octanoic acid and sodium octanoate; the molar ratio of n-octanoic acid to sodium octanoate is 2-3:1.

[0006] In the above-mentioned deep eutectic solvent, the molar ratio of n-octanoic acid to sodium octanoate is 2:1.

[0007] The present invention also provides the use of the deep eutectic solvent in the simultaneous extraction of water-soluble and water-insoluble edible fungus polysaccharides.

[0008] In the above application, the edible fungus is golden ear.

[0009] Furthermore, the present invention provides a method for extracting polysaccharides from edible fungi, comprising the following steps:

[0010] (1) adjusting the water content and pH value of the deep eutectic solvent to obtain DES1; the pH value of DES1 is 11-13;

[0011] (2) mixing the edible fungi and the DES1, heating extraction to obtain a precipitate 1 and a supernatant; adding anhydrous ethanol to the supernatant, and the obtained precipitate is a water-soluble polysaccharide;

[0012] (3) adjusting the water content and pH value of the DES to obtain DES2; the pH value of the DES2 is 1-3;

[0013] (4) mixing the precipitate 1 obtained in step (2) and the DES2, heating extraction, and taking the supernatant; adding anhydrous ethanol to the supernatant, and the obtained precipitate is a water-insoluble polysaccharide. In the above method, in step (1), the volume percentage water content of the DES1 is 82%-98%; preferably 84%-88%; more preferably 84% or 88%.

[0014] In step (3), the volume percentage water content of the DES2 is 10%-90%; preferably 20%-40%; more preferably 40%.

[0015] In the above method, the edible fungi are edible fungi powder; specifically, the edible fungi can be crushed and passed through a 100-mesh sieve.

[0016] In the above method, in step (2), the solid-liquid ratio of the edible fungi and the DES1 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; more preferably 1:42.5 or 1:47.5.

[0017] The heating extraction temperature is 95-100°C; specifically, it can be 100°C.

[0018] The heating extraction time is 0.5-2.5 h; preferably 1.75-2.25 h; more preferably 2.25 h.

[0019] In the above method, in step (3), the deep eutectic solvent can be obtained by rotary evaporation of the supernatant after alcohol precipitation in step (2).

[0020] In the above method, in step (4), the solid-liquid ratio of the precipitate 1 and the DES2 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; specifically, it can be 1:42.5.

[0021] The heating extraction temperature is 95-100°C; specifically, it can be 100°C.

[0022] The heating extraction time is 0.5-2.5 h; preferably 1.75-2.25 h; specifically, it can be 1.75 h or 2.25 h.

[0023] In the above method, in steps (2) and (4), the volume ratio of the supernatant to anhydrous ethanol is 1:4-5, specifically 1:4.

[0024] In the above method, in steps (2) and (4), the supernatant is added with anhydrous ethanol and then allowed to stand; specifically, it is allowed to stand at 4°C overnight, more specifically, it can be allowed to stand at 4°C for 12 hours.

[0025] The above method, step (2) further comprises the steps of re-dissolving the precipitate obtained after adding anhydrous ethanol with water and freeze-drying;

[0026] Step (4) also includes a step of freeze-drying the precipitate obtained after adding anhydrous ethanol.

[0027] The present invention has the following advantages:

[0028] (1) This method can achieve the hydrophilic-hydrophobic switching of the low eutectic solvent by regulating the pH value of the extraction system, and simultaneously extract water-soluble and water-insoluble polysaccharides.

[0029] (2) This method has a high yield of polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The graph shows the DES mixing results of different molar ratios (octanoic acid and sodium octanoate).

[0031] Figure 2 The effect of different molar ratios on the apparent viscosity of DES.

[0032] Figure 3 This is the flow chart of Tremella fuciformis polysaccharide extraction.

[0033] Figure 4 The effect of different material-liquid ratios on polysaccharide yield.

[0034] Figure 5 The effect of different extraction times on the polysaccharide yield.

[0035] Figure 6 The effect of different DES water content on polysaccharide yield.

[0036] Figure 7 These are scanning electron microscope images of the water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in Example 3.

[0037] Figure 8 This is the infrared spectrum of the water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in Example 3. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0039] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0040] The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0041] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0042] The materials and instruments used in the following examples are as follows:

[0043] Sodium octanoate (purity 99%); n-octanoic acid (purity 99%); anhydrous ethanol. The reagents used are all of analytical purity; the laboratory water is all distilled water.

[0044] Digital constant temperature water bath, Shanghai Boxin Co., Ltd.; high-speed refrigerated centrifuge, Thermo Fisher Scientific China Co., Ltd.; rotary evaporator, Germany IKA Company; pH meter, Jinan Laibao Medical Instrument Co., Ltd.; vacuum freeze dryer, Germany MarinChrist Company.

[0045] Example 1, preparation of deep eutectic solvent (DES)

[0046] The n-octanoic acid and sodium octanoate were mixed according to n(C8H 16 O2):n(C8H 15 NaO2) as 3:1, 2:1, 1:1, 1:2, 1:3 at 80°C, and the density and rheological properties of the system that could form a stable and uniform system were determined, and the appropriate ratio was selected for subsequent experiments.

[0047] The results of the five combinations are shown in Figure 1 O2):n(C8H 16 O2):n(C8H 15 NaO2) as 3:1 and 2:1 can form transparent and stable solutions, so these two ratios were selected for subsequent experiments. The static rheological results are shown in Figure 2 As the shear rate increases, the viscosity of both is relatively stable, and the viscosity of the DES with a molar ratio of 2:1 is higher. The density results are shown in Table 1, and the difference in density between the two is not obvious. Through the pre-experiment, the yield of polysaccharide of the DES with a molar ratio of 2:1 is higher, so this ratio is selected for subsequent experiments.

[0048] Table 1 Density determination table of two DESs

[0049]

[0050] According to the above results, n(C8H 16 O2):n(C8H15 NaO2) = 2:1 and then stirred continuously at 80 ° C until a transparent and stable liquid is formed, which is named deep eutectic solvent (DES); the obtained DES is stored in a dry and cool place for future use.

[0051] Example 2, Tremella polysaccharide ( Naematelia aurantialba Extraction of polysaccharides, NAP

[0052] The extraction method of NAP is as follows Figure 3 As shown. Add appropriate amount of golden fungus ( Naematelia aurantialba, NA) was dried and pulverized, passed through a 100-mesh sieve, and the powder was collected for later use. The pH of DES was adjusted to 12 using a NaOH solution, and distilled water was added to prepare DES1 with water contents of 82, 86, 90, 94, and 98% (volume percentage) for later use. 1 g of NA was added to DES1 at a material-to-liquid ratio of 1:30-50 (g:mL). The mixture was heated in a 100°C waterbath for 0.5-2.5 hours, followed by centrifugation at 8500 rpm for 10 minutes. The supernatant was collected and designated as Precipitate 1. Anhydrous ethanol was added to the supernatant at a V (supernatant) to V (anhydrous ethanol) ratio of 1:4 (volume ratio). The mixture was allowed to stand for 12 hours before centrifugation. The precipitate was reconstituted with distilled water and freeze-dried to obtain water-soluble Naematelia aurantialba polysaccharides (NAP-S). The supernatant after alcohol precipitation was rotary evaporated to collect DES, and its pH was adjusted to 2 with 3 mol / L HCl solution. Distilled water was added to it to prepare DES2 with different water contents (10, 30, 50, 70, and 90%). DES2 was added to precipitate 1 according to the same material-liquid ratio as NA and DES1, and the mixture was heated in a 100°C water bath for the same extraction time as water-soluble Tremella fuciformis polysaccharides. Subsequently, the mixture was centrifuged at 8500 r / min for 10 min, and the supernatant was collected. Anhydrous ethanol was added according to V (supernatant): V (anhydrous ethanol) = 1:4 (volume ratio). After standing for 12 h, the mixture was centrifuged and the precipitate was freeze-dried to obtain water-insoluble Naematelia aurantialba polysaccharides (NAP-I).

[0053] 1. Single-factor experiment

[0054] The yields of NAP-S and NAP-I were used as indicators. The effects of different solid-liquid ratios (1:30, 1:35, 1:40, 1:45, and 1:50 g / mL) on polysaccharide yield were investigated, with an extraction time of 1.5 h and water contents of DES1 and DES2 of 90% and 50%, respectively. The effects of extraction time (0.5, 1, 1.5, 2, and 2.5 h) on polysaccharide yield were investigated, with a solid-liquid ratio of 1:45 and water contents of DES1 and DES2 of 90% and 50%, respectively. The effects of different DES1 and DES2 water content combinations (82% / 10%, 86% / 30%, 90% / 50%, 94% / 70%, and 98% / 90%) on polysaccharide yield were investigated, with an extraction time of 2 h and a solid-liquid ratio of 1:45.

[0055]

[0056] Where: m1 is the mass of the freeze-dried polysaccharide sample; m2 is the mass of the golden ear used; x is the polysaccharide yield.

[0057] (1) Effect of material-liquid ratio on the yield of Tremella fuciformis polysaccharide

[0058] Effects of different material-liquid ratios on the yield of Tremella fuciformis polysaccharide Figure 4 As shown, as the solid-liquid ratio increases, the polysaccharide yield initially increases and then decreases. The yield of both polysaccharides is highest at a solid-liquid ratio of 1:45. The increase in polysaccharide yield may be due to the increased contact area between DES and NA, resulting in higher sugar extraction efficiency. The subsequent decrease in polysaccharide yield may be due to the weakening of hydrogen bonding between DES and NAP, which reduces extraction efficiency. Therefore, 1:45 was selected as the optimal solid-liquid ratio.

[0059] (2) Effect of extraction time on the yield of Tremella fuciformis polysaccharides

[0060] As the extraction time increases, DES can fully extract the polysaccharides in NA, thereby increasing the yield. However, the longer the extraction time, not only will it fail to increase the NAP content, but it will also destroy the polysaccharides that have been produced, thereby reducing the polysaccharide yield ( Figure 5 ). According to the total sugar yield, 2 h was selected as the optimal extraction time.

[0061] (3) Effect of water content on the yield of Tremella fuciformis polysaccharide

[0062] Effect of water content on polysaccharide yield Figure 6The DES itself has high viscosity, so changing the water content of DES can change the viscosity and surface tension of DES, promoting polysaccharide extraction. When the water content combination is 86% / 30%, the yield of NAP-S is the highest, and when the water content combination is 90% / 50%, the yield of NAP-I is the highest. The yield decreases when the water content is too high, which may be because water weakens the interaction between DES and NAP. Therefore, according to the total sugar yield, 86% / 30% is selected as the optimal water content of DES1 and DES2.

[0063] 2. Orthogonal test design

[0064] To determine the optimal extraction process, based on the results of single-factor experiments, factors A (solid-liquid ratio), B (extraction time), and C (DES water content) were selected, and the yields of NAP-S and NAP-I were used as indicators to design a three-factor three-level experiment (Table 2) to further optimize the process of extracting gold ear polysaccharide with DES. The optimal parameters were used to prepare NAP-S / I.

[0065] Table 2 Orthogonal test design table

[0066]

[0067] Note: In the water content combination, 84, 86, and 88 refer to the water content of DES1, and 20, 30, and 40 refer to the water content of DES2.

[0068] A standard curve was prepared using the phenol-sulfuric acid method, with absorbance as the vertical coordinate and concentration as the horizontal coordinate. The regression equation was y = 3.4526x - 0.0557. A 1 mg / mL polysaccharide solution was prepared, and 200 μL of the polysaccharide solution was taken and added to 200 μL of 5% phenol by volume percentage and 1 mL of concentrated sulfuric acid. The mixture was placed in a 30°C metal bath for 30 min, and the absorbance was measured at 490 nm. The test was repeated three times.

[0069] The optimal DES extraction process was obtained by single factor experiment, and the ratio of material to liquid was 1:45, the extraction time was 2 h, and the water content of DES was 86% / 30%. The orthogonal optimization results of DES extraction of NAP are shown in Table 3. The order of the influence of each index on the extraction efficiency of NAP was C>A>B (NAP-S) and A>C>B (NAP-I), that is, the water content of DES>the ratio of material to liquid>the extraction time (NAP-S), the ratio of material to liquid>the water content of DES>the extraction time (NAP-I). The highest total polysaccharide yield was 73.1%. The optimal scheme for extracting NAP-S was that the ratio of material to liquid was 1:47.5, the extraction time was 2.25 h, and the water content of DES1 was 84%. The optimal scheme for extracting NAP-I was that the ratio of material to liquid was 1:42.5, the extraction time was 1.75 h, and the water content of DES2 was 40%. The optimal scheme for extracting NAP-S and NAP-I was that the ratio of material to liquid was 1:42.5, the extraction time was 2.25 h, the water content of DES1 was 88%, and the water content of DES2 was 40%.

[0070] Table 3 Orthogonal test results

[0071]

[0072] Example 3

[0073] An appropriate amount of NA was dried and crushed, and then passed through a 100-mesh sieve to collect the powder for use. The pH of DES was adjusted to 12 with a NaOH solution, and then distilled water was added to prepare a DES1 with a water content of 84% (volume percentage) for storage. 1 g of NA was added to DES1 according to a ratio of 1:47.5 (g:mL), and then heated in a water bath at 100°C for 2.25 h. Subsequently, centrifugation was performed at 8500 r / min for 10 min, and the supernatant was collected, and the precipitate was named as precipitate 1. The supernatant was added with anhydrous ethanol according to a volume ratio of V(supernatant):V(anhydrous ethanol)=1:4, and then centrifugation was performed after being placed for 12 h. The precipitate was redissolved with distilled water and then freeze-dried to obtain water-soluble golden ear polysaccharide (NAP-S). The supernatant after alcohol precipitation was rotary evaporated to collect DES, and then 3 mol / L HCl solution was added to adjust the pH to 2. Distilled water was added to prepare a DES2 with a water content of 40%. According to a ratio of 1:42.5 (g:mL), DES2 was added, and then heated in a water bath at 100°C for 1.75 h. Subsequently, centrifugation was performed at 8500 r / min for 10 min, and the supernatant was collected. Anhydrous ethanol was added according to a volume ratio of V(supernatant):V(anhydrous ethanol)=1:4, and then centrifugation was performed after being placed for 12 h. The precipitate was freeze-dried to obtain water-insoluble golden ear polysaccharide (NAP-I). The purity of NAP-S and NAP-I extracted by the above method was 72.86% and 64.38%, respectively.

[0074] Table 4 Monosaccharide composition of the extracted polysaccharide

[0075]

[0076] Table 5 Molecular weight of extracted polysaccharides

[0077]

[0078] The water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in this example were observed by scanning electron microscopy. Figure 7 ,Depend on Figure 7 As can be seen from the microscopic appearance of NAP at magnifications of ×100 and ×300, NAP-I is generally flat, large, and smooth. NAP-S is generally more fragmented, with cavities of varying sizes and a small amount of fiber on the surface.

[0079] The infrared spectra of the water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in this example are shown in FIG. Figure 8 ,Depend on Figure 8 It can be seen that at 3400 cm -1 A strong and broad absorption band is generated at the left and right sides, which is caused by the stretching vibration of OH or CH. This absorption band is a characteristic absorption band of polysaccharides. -1 and 1722cm -1 The stretching vibration of C=O was detected, among which the absorption band of NAP-S had stronger vibration.

[0080] The monosaccharide compositions of the water-soluble and water-insoluble Tremella fuciformis polysaccharides extracted in this example are shown in Table 4. As shown in Table 4, NAP-S and NAP-I are primarily composed of mannose, glucose, and xylose. However, the molar ratios of the constituent monosaccharides differ; NAP-I contains fucose, but NAP-S does not.

[0081] The molecular weights of the water-soluble and water-insoluble Tremella fuciformis polysaccharides extracted in this example are shown in Table 5. As shown in Table 5, multiple peaks appear in NAP-S, indicating that its molecular weight distribution is relatively non-uniform. The molecular weight of NAP-S is significantly higher than that of NAP-I.

Claims

1. A method for extracting polysaccharides from edible fungi, characterized by: The deep eutectic solvent used is composed of n-octanoic acid and sodium octanoate; the molar ratio of n-octanoic acid to sodium octanoate is 2-3:1; The method comprises the following steps: (1) adjusting the water content and pH value of the deep eutectic solvent to obtain DES1; the pH value of DES1 is 11-13; (2) mixing the edible fungus and the DES1, heating and extracting the mixture to obtain a precipitate 1 and a supernatant; adding anhydrous ethanol to the supernatant to obtain a precipitate that is a water-soluble polysaccharide; (3) adjusting the water content and pH value of the deep eutectic solvent to obtain DES2; the pH value of DES2 is 1-3; (4) The precipitate 1 obtained in step (2) and the DES2 are mixed, heated for extraction, and the supernatant is obtained; anhydrous ethanol is added to the supernatant, and the resulting precipitate is a water-insoluble polysaccharide.

2. The method according to claim 1, wherein: The molar ratio of n-octanoic acid to sodium octanoate is 2:

1.

3. The method according to claim 1, wherein: The edible fungus is golden ear.

4. The method according to claim 1, wherein: In step (1), the volume percentage water content of DES1 is 82%-98%; In step (3), the volume percentage water content of the DES2 is 10%-90%.

5. The method according to claim 4, characterized in that: In step (1), the volume percentage water content of DES1 is 84%-88%; In step (3), the volume percentage water content of the DES2 is 20%-40%.

6. The method according to claim 1, wherein: In step (2), the material-liquid ratio of the edible fungus and DES1 is 1 g:30-50 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h.

7. The method according to claim 6, wherein: In step (2), the material-liquid ratio of the edible fungus and DES1 is 1 g:42.5-47.5 mL; The heating extraction time is 1.75-2.25 h.

8. The method according to claim 1, wherein: In step (4), the material-liquid ratio of the precipitate 1 and DES2 is 1 g:30-50 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5 h.

9. The method according to claim 8, wherein: In step (4), the material-liquid ratio of the precipitate 1 and DES2 is 1 g:42.5-47.5 mL; The heating extraction time is 1.75-2.25 h.

10. The method according to claim 1, wherein: In steps (2) and (4), the volume ratio of the supernatant to anhydrous ethanol is 1:4-5.

11. The method according to claim 1, wherein: Step (2) also includes the steps of re-dissolving the precipitate obtained after adding anhydrous ethanol with water and freeze-drying; Step (4) also includes a step of freeze-drying the precipitate obtained after adding anhydrous ethanol.

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