Eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible mushroom polysaccharides and application of eutectic solvent

Abstract

The invention discloses a deep-eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible mushroom polysaccharides and application of the deep-eutectic solvent, and belongs to the technical field of natural product extraction. The deep-eutectic solvent for simultaneously extracting the water-soluble and water-insoluble edible mushroom polysaccharides is composed of n-caprylic acid and sodium caprylate; the molar ratio of n-caprylic acid to sodium caprylate is (2-3): 1. According to the method disclosed by the invention, the pH value of an extraction system can be regulated and controlled to realize hydrophilic-hydrophobic switching of the eutectic solvent, and water-soluble and non-water-soluble polysaccharides are extracted at the same time; and the yield of the extracted polysaccharide is high.

Description

Technical Field

[0001] The 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 application thereof. Background Art

[0002] Naematelia aurantialba (NA) belongs to the Tremellales, Ototrichum family, Ototrichum genus, and is parasitic on the fungus. The fruiting body of Naematelia aurantialba is golden in appearance, shaped like a human brain, and is golden yellow. As a precious edible and medicinal fungus, it is rich in nutrients, mainly including polysaccharides, fats, minerals, carotenoids, etc. Among them, polysaccharides are one of the most important bioactive ingredients in Naematelia aurantialba, which has good antioxidant, hypoglycemic, hypolipidemic, and anti-inflammatory functions.

[0003] Common extraction solvents for natural polysaccharides include water, acid, alkali, etc. The yield of polysaccharides extracted by these traditional solvents is generally low. In recent years, deep eutectic solvent (DES) has been widely used as a new type of green solvent. It is a mixture of hydrogen bond acceptors and hydrogen bond donors in a specific ratio, and its properties are similar to ionic liquids. At present, there have been reports on the application of DES in polysaccharide extraction, but DES is only used to replace traditional solvents to extract water-soluble polysaccharides, and the effective extraction and separation of hydrophilic and hydrophobic active substances has not been achieved by making full use of the hydrophilic and hydrophobic state of DES. Summary of the invention

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

[0005] The invention firstly provides a low eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible fungus polysaccharides, which 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 low 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 low eutectic solvent in the simultaneous extraction of water-soluble and water-insoluble edible fungus polysaccharides.

[0008] In the above application, the edible fungus is Acanthopanax edulis.

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

[0010] (1) adjusting the water content and pH value of the deep eutectic solvent according to claim 1 or 2 to obtain DES1; the pH value of DES1 is 11-13;

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

[0012] (3) adjusting the water content and pH value of the deep eutectic solvent according to claim 1 or 2 to obtain DES2; the pH value of DES2 is 1-3;

[0013] (4) Mixing the precipitate 1 obtained in step (2) and the DES2, heating and extracting, and taking the supernatant; adding anhydrous ethanol to the supernatant, and the resulting 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 DES2 is 10%-90%, preferably 20%-40%, and more preferably 40%.

[0015] In the above method, the edible fungus is edible fungus powder; specifically, it can be obtained by crushing the edible fungus and then passing it through a 100-mesh sieve.

[0016] In the above method, in step (2), the material-liquid ratio of the edible fungus to 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 temperature of the heating extraction is 95-100°C, and can be specifically 100°C;

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

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

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

[0021] The temperature of the heating extraction is 95-100°C, and can be specifically 100°C;

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

[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] In 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 adjusting the pH value of the extraction system, thereby extracting both water-soluble and water-insoluble polysaccharides.

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

[0030] Figure 1 This is a graph showing 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 solid-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 This is a scanning electron microscope image 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 spectra of the water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in Example 3. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but 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 were all repeated three times, and the results were averaged.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[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. All reagents used were analytical grade; and the laboratory water was distilled water.

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

[0045] Example 1. Preparation of Deep Eutectic Solvent (DES)

[0046] Octanoic acid and sodium octanoate were mixed according to n(C8H 16 O2):n(C8H 15 NaO2) in the ratio of 3:1, 2:1, 1:1, 1:2, 1:3 and heated at 80°C. Select the system that can form a stable and uniform system to measure the density and rheological properties, and choose the appropriate ratio for subsequent tests.

[0047] The results of the five combinations are as follows Figure 1 As shown, only when n(C8H 16 O2):n(C8H 15 When the ratio of NaO2 is 3:1 or 2:1, a transparent and stable solution can be formed. Therefore, these two ratios are selected for subsequent tests. Figure 2 As shown in Table 1, as the shear rate increases, the viscosity of both is relatively stable, and the viscosity of DES with a molar ratio of 2:1 is higher. The density results are shown in Table 1, and the density difference between the two is not obvious. Through preliminary experiments, the polysaccharide yield of 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 DES

[0049] <![CDATA[n(C8H 16 O2:C8H 15 NaO2)]]> 3:1 2:1 <![CDATA[Density / g·cm -3 > 0.97 0.94

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

[0051] Example 2: Extraction of Naematelia aurantialba polysaccharides (NAP)

[0052] The extraction method of NAP is as follows Figure 3 As shown. Dry and crush an appropriate amount of Naematelia aurantialba (NA), pass through a 100-mesh sieve, and collect the powder for later use. Adjust the pH of DES to 12 with NaOH solution, add distilled water to prepare DES1 with a water content of 82, 86, 90, 94, and 98% (volume percentage) for later use. Take 1g NA and add DES1 at a solid-liquid ratio of 1:30-50 (g:mL), heat and extract in a 100℃ water bath for 0.5-2.5h, then centrifuge at 8500r / min for 10min, take the supernatant, and name the precipitate 1. Add anhydrous ethanol to the supernatant according to V (supernatant): V (anhydrous ethanol) = 1:4 (volume ratio), centrifuge after standing for 12h; reconstitute the precipitate with distilled water and freeze-dry to obtain water-soluble Naematelia aurantialba polysaccharides (NAP-S). The supernatant after alcohol precipitation was evaporated, DES was collected, and its pH was adjusted to 2 with 3 mol / L HCl solution, distilled water was added thereto to prepare DES2 with different water contents (10, 30, 50, 70, 90%), DES2 was added to precipitate 1 according to the same solid-liquid ratio as NA and DES1, and the extraction was heated in a 100°C water bath for the same extraction time as water-soluble Tremella aurantialba polysaccharides, followed by centrifugation at 8500r / min for 10min, the supernatant was taken, and anhydrous ethanol was added according to V (supernatant): V (anhydrous ethanol) = 1:4 (volume ratio), and the mixture was allowed to stand for 12h and then centrifuged, and the precipitate was freeze-dried to obtain water-insoluble Naematelia aurantialba polysaccharides (NAP-I).

[0053] 1. Single Factor Experiment

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

[0055]

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

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

[0058] Effects of different solid-liquid ratios on the yield of Tremella fuciformis polysaccharides Figure 4 As shown in the figure, as the solid-liquid ratio increases, the polysaccharide yield increases first and then decreases. When the solid-liquid ratio is 1:45, the yields of both polysaccharides are the highest. The increase in polysaccharide yield may be due to the increase in the contact area between DES and NA, which leads to a higher sugar extraction efficiency. The subsequent decrease in polysaccharide yield may be due to the weakening of the hydrogen bond between DES and NAP, which reduces the extraction efficiency. Therefore, 1:45 is 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, 2h 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 6As shown. DES itself has a high viscosity, so changing the water content of DES can change the viscosity and surface tension of DES and promote 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 decrease in yield when the water content is too high may be because the water weakens the interaction between DES and NAP. Therefore, according to the total sugar yield, 86% / 30% was selected as the optimal water content of DES1 and DES2.

[0063] 2. Orthogonal Experimental Design

[0064] In order to clarify the optimal extraction process, based on the results of single-factor experiments, A (solid-liquid ratio), B (extraction time), and C (DES water content) were used as factors, and the yields of NAP-S and NAP-I were used as indicators. Three factors and three levels were designed for experiments (Table 2) to further optimize the DES extraction process of Tremella polysaccharides. The optimal parameters were selected to prepare NAP-S / I.

[0065] Table 2 Orthogonal test design table

[0066]

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

[0068] The standard curve was prepared by the phenol-sulfuric acid method, with absorbance as the ordinate and concentration as the abscissa. The regression equation was y = 3.4526x-0.0557. Prepare 1 mg / mL polysaccharide solution, take 200 μL of polysaccharide solution, add 200 μL of 5% phenol and 1 mL of concentrated sulfuric acid, place in a 30°C metal bath for 30 minutes, and measure the absorbance at 490 nm. Repeat the experiment three times.

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

[0070] Table 3 Orthogonal test results

[0071]

[0072] Example 3

[0073] Dry and crush an appropriate amount of NA, pass it through a 100-mesh sieve, and collect the powder for later use. Adjust the pH of DES to 12 with NaOH solution, add distilled water to prepare DES1 with a water content of 84% (volume percentage) and store it for later use. Take 1g of NA and add it to DES1 at a solid-liquid ratio of 1:47.5 (g:mL), heat and extract it in a 100℃ water bath for 2.25h, then centrifuge it at 8500r / min for 10min, take the supernatant, and name the precipitate 1. Add anhydrous ethanol to the supernatant according to V (supernatant): V (anhydrous ethanol) = 1:4 (volume ratio), centrifuge it after standing for 12h; reconstitute the precipitate with distilled water and freeze-dry it to obtain water-soluble Tremella polysaccharide (NAP-S). The supernatant after alcohol precipitation was evaporated, DES was collected, and its pH was adjusted to 2 with 3 mol / L HCl solution, distilled water was added thereto, and DES2 with a water content of 40% was prepared, and DES2 was added according to a solid-liquid ratio of 1:42.5 (g:mL), and heated in a 100°C water bath for extraction for 1.75h, followed by centrifugation at 8500r / min for 10min, and the supernatant was taken, and anhydrous ethanol was added according to V (supernatant): V (anhydrous ethanol) = 1:4 (volume ratio), and centrifuged after standing for 12h, and the precipitate was freeze-dried to obtain water-insoluble Tremella polysaccharide (NAP-I). The purities of NAP-S and NAP-I polysaccharides extracted by the above method were 72.86% and 64.38%, respectively.

[0074] Table 4 Monosaccharide composition of the extracted polysaccharides

[0075]

[0076] Table 5 Molecular weight of extracted polysaccharides

[0077]

[0078] The scanning electron microscopy of the water-soluble Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in this example shows Figure 7 ,Depend on Figure 7 It can be seen that the microscopic appearance of NAP is magnified at ×100 and ×300. NAP-I is generally flat, large and smooth. NAP-S is generally broken, with holes of varying sizes and a small amount of fiber filaments on the surface.

[0079] The infrared spectra of the water-soluble Tremella fuciformis polysaccharides and water-insoluble Tremella fuciformis polysaccharides extracted in this example are shown in FIG. Figure 8 ,Depend on Figure 8 It can be seen that at 3400cm -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 Tremella fuciformis polysaccharide and the water-insoluble Tremella fuciformis polysaccharide extracted in this example are shown in Table 4. As can be seen from Table 4, NAP-S and NAP-I are mainly composed of mannose, glucose, and xylose. However, there are differences in the molar ratio of the constituent monosaccharides, and NAP-I contains fucose, but NAP-S does not contain this monosaccharide.

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

Claims

1. A low eutectic solvent for simultaneously extracting water-soluble and water-insoluble edible fungus polysaccharides, which is composed of n-octanoic acid and sodium octanoate; the molar ratio of n-octanoic acid to sodium octanoate is 2-3:

1.

2. The deep eutectic solvent according to claim 1, characterized in that: The molar ratio of n-octanoic acid to sodium octanoate is 2:

1.

3. Use of the deep eutectic solvent according to claim 1 or 2 in the simultaneous extraction of water-soluble and water-insoluble edible fungus polysaccharides.

4. The use according to claim 3, characterized in that: The edible fungus is golden ear.

5. A method for extracting edible fungus polysaccharides, comprising the following steps: (1) adjusting the water content and pH value of the deep eutectic solvent according to claim 1 or 2 to obtain DES1; the pH value of DES1 is 11-13; (2) mixing the edible fungus and the DES1, heating and extracting, to obtain a precipitate 1 and a supernatant; adding anhydrous ethanol to the supernatant, and obtaining a precipitate that is a water-soluble polysaccharide; (3) adjusting the water content and pH value of the deep eutectic solvent according to claim 1 or 2 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 obtained precipitate is a water-insoluble polysaccharide.

6. The method according to claim 5, characterized in that: In step (1), the volume percentage water content of DES1 is 82%-98%; preferably 84%-88%; In step (3), the volume percentage water content of DES2 is 10%-90%, preferably 20%-40%.

7. The method according to claim 5 or 6, characterized in that: In step (2), the material-liquid ratio of the edible fungus to DES1 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5h; preferably 1.75-2.25h.

8. The method according to any one of claims 5 to 7, characterized in that: In step (4), the solid-liquid ratio of the precipitate 1 to DES2 is 1 g:30-50 mL; preferably 1 g:42.5-47.5 mL; The temperature of the heating extraction is 95-100°C; The heating extraction time is 0.5-2.5h; preferably 1.75-2.25h.

9. The method according to any one of claims 5 to 8, characterized in that: In steps (2) and (4), the volume ratio of the supernatant to anhydrous ethanol is 1:4-5.

10. The method according to any one of claims 5 to 9, characterized in that: 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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