Dual-frequency ultrasonic-assisted extraction method of dioscorea polysaccharide and application

Abstract

The application discloses a double-frequency ultrasonic-assisted extraction method of yam polysaccharide and application, and belongs to the technical field of natural product extraction. The method comprises the following steps: S1, preparing an ethanol solution of yam powder; S2, placing the solution in a double-frequency ultrasonic device for ultrasonic-assisted extraction, and the ultrasonic conditions comprise the following: an ultrasonic frequency of 40 kHz and 25 kHz, an ultrasonic power of 345-525 W, and an ultrasonic time of 4-12 min; and S3, filtering to obtain yam polysaccharide extract. The method is an efficient, rapid and simple yam polysaccharide extraction method, is particularly suitable for an industrialized production scene with strict requirements on extraction time, and the extracted yam polysaccharide has good in-vitro antioxidant activity, can be used for preparing antioxidant-related functional food, medicine and cosmetics, and has a good development prospect in the field of health products.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a dual-frequency ultrasound-assisted extraction method for yam polysaccharides and its application. Background Technology

[0002] Yam (Dioscorea opposita Thunb.), also known as Chinese yam, is the dried rhizome of the Dioscorea opposita plant, belonging to the Dioscoreaceae family. It is a traditional Chinese plant used for both food and medicine, widely distributed throughout northern and southern China, with a long history of edible and medicinal use. According to the *Compendium of Materia Medica*, yam is warm in nature and sweet in taste, entering the spleen, stomach, and lung meridians, and possessing various effects such as strengthening the spleen and stomach, tonifying the lungs and kidneys, and nourishing and strengthening the body. Yam contains various bioactive components, including polysaccharides, mucin, starch, amino acids, and phenolic compounds. Among them, yam polysaccharides are one of the most important active components of yam, belonging to natural high-molecular-weight carbohydrates with good biocompatibility and safety. Recent studies have shown that yam polysaccharides have various biological activities, such as antioxidant, hypoglycemic, anti-fatigue, and immunomodulatory effects. Its antioxidant and hypoglycemic effects are particularly significant, playing an important role in scavenging free radicals, increasing insulin sensitivity and insulin secretion, and improving the body's disease resistance. Therefore, yam polysaccharides have significant research and development value in the fields of functional foods, pharmaceuticals, and cosmetics.

[0003] While traditional hot water extraction is simple to operate, it suffers from drawbacks such as long extraction time, high energy consumption, polysaccharide degradation, and loss of activity. In contrast, ultrasound-assisted extraction, as a green and efficient method, has attracted widespread attention in recent years. This method utilizes the cavitation, mechanical, and thermal effects generated by ultrasound propagation in liquids to effectively disrupt cell wall structures, promote full contact between the solvent and raw material, thereby improving extraction efficiency and polysaccharide yield, shortening extraction time, reducing energy consumption, and helping to preserve the natural structure and bioactivity of polysaccharides. However, traditional single-frequency ultrasound extraction still has limitations, such as uneven sound field energy distribution, the tendency to form standing waves leading to incomplete cavitation, and the need for further improvement in extraction efficiency and stability. Dual-frequency ultrasound-assisted extraction is a novel extraction method that avoids the problems of uneven sound field, incomplete cavitation, and standing waves affecting the extraction rate associated with single-frequency ultrasound, and thus has significant development potential. However, research on systematic dual-frequency ultrasonic extraction technology for yam polysaccharides is still lacking, especially in optimizing key parameters, where a mature, efficient, and reproducible process has not yet been developed, hindering the application of this technology in yam polysaccharide extraction. Therefore, developing an efficient, stable, and optimized method for yam polysaccharide extraction based on dual-frequency ultrasound assistance is of great significance for enhancing the comprehensive utilization value of yam resources and promoting the development of related functional food industries. Summary of the Invention

[0004] This invention provides a dual-frequency ultrasound-assisted extraction method for yam polysaccharides and its application, aiming to solve the problems of the prior art.

[0005] The dual-frequency ultrasound-assisted extraction method for yam polysaccharides described in this invention includes the following steps: S1. Prepare an ethanol solution of yam powder; S2. Place the above solution in a dual-frequency ultrasonic device for ultrasonic-assisted extraction. The ultrasonic conditions include: ultrasonic frequency of 40kHz and 25kHz, ultrasonic power of 345-525W, and ultrasonic time of 4-12min. S3. After filtration, yam polysaccharide extract is obtained.

[0006] Specifically, in step S1, the liquid-to-solid ratio of yam powder to ethanol is 10-50 mL / g; preferably, the liquid-to-solid ratio is 25-40 mL / g; and most preferably, the liquid-to-solid ratio is 32.5 mL / g.

[0007] Specifically, in step S2, preferably, the ultrasonic power is 390-480W; most preferably, the ultrasonic power is 435W.

[0008] Specifically, in step S2, preferably, the ultrasound time is 8-12 min; most preferably, the ultrasound time is 10.7 min.

[0009] The present invention also provides a yam polysaccharide extracted by the above method, which exhibits significant activity in in vitro antioxidant activity tests and has the potential to be used as a natural antioxidant.

[0010] This invention also provides an application of the above-mentioned yam polysaccharide as an antioxidant, which can be used to prepare antioxidant-related functional foods, pharmaceuticals and cosmetics, and has good development prospects in the field of health products.

[0011] The beneficial effects of this invention are as follows: 1. This invention employs a specific dual-frequency synergistic and optimized extraction process combination, which can complete efficient extraction within 10 minutes. The time required is only 1 / 12 of that of the traditional hot water extraction method and about 1 / 9 of that of the conventional single-frequency ultrasonic method, which greatly improves production efficiency and equipment utilization.

[0012] 2. The method of the present invention does not require the addition of chemical reagents or biological enzymes, does not rely on complex subsequent separation steps, has a simple process route, is easy to control, is more in line with the requirements of green production, and is suitable for large-scale production.

[0013] 3. The yam polysaccharide extracted by the method of the present invention has been shown to have significant in vitro antioxidant activity, proving that the method of the present invention can effectively preserve the biological activity of natural products, providing a good application basis for its application in functional foods, pharmaceuticals and cosmetics. Attached Figure Description

[0014] Figure 1 : Glucose standard curve.

[0015] Figure 2 Figure showing the effect of ultrasound time on the extraction rate of yam polysaccharides.

[0016] Figure 3 Figure: Effect of liquid-to-solid ratio on the extraction rate of yam polysaccharides.

[0017] Figure 4 Figure: Effect of ultrasonic power on the extraction rate of yam polysaccharides.

[0018] Figure 5 Contour plot (A) and 3D response surface plot (B) showing the effects of ultrasonic time and liquid-to-solid ratio on extraction rate; Contour plot (C) and 3D response surface plot (D) showing the effects of ultrasonic time and ultrasonic power on extraction rate; Contour plot (E) and 3D response surface plot (F) showing the effects of liquid-to-solid ratio and ultrasonic power on extraction rate.

[0019] Figure 6 Figure: DPPH free radical scavenging test results of yam polysaccharides.

[0020] Figure 7 Figure: ABTS free radical scavenging test results of yam polysaccharides.

[0021] Figure 8 Figure: Results of hydroxyl radical scavenging test of yam polysaccharides.

[0022] Figure 9 Figure: Results of iron reducing power test for yam polysaccharides. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments.

[0024] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0025] When a mass, concentration, temperature, time, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, it should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, the range 1-50 should be understood to include any number, combination of numbers, or subranges selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all decimal values ​​between the integers mentioned above, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Regarding subranges, specifically consider "nested subranges" extending from any endpoint of the range. For example, nested sub-ranges of the exemplary range 1-50 may include 1-10, 1-20, 1-30 and 1-40 in one direction, or 50-40, 50-30, 50-20 and 50-10 in another direction.

[0026] The singular form includes the plural objects of discussion unless the context clearly indicates otherwise. "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event occurs and the possibility that the event does not occur.

[0027] Approximate terms used in the specification and claims to modify quantities indicate that the invention is not limited to that specific quantity, but also includes acceptable modifications close to that quantity that do not alter the relevant essential function. Correspondingly, the use of "about," "approximately," etc., to modify a numerical value means that the invention is not limited to that precise value. In some instances, approximate terms may correspond to the precision of the instrument used to measure the value. In this application's specification and claims, scope definitions can be combined and / or interchanged, unless otherwise stated, these scopes include all subscopes contained therein.

[0028] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of this invention do not impose any limitation on the quantity requirement (i.e., the number of times) of the elements or components. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers to the singular form.

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] In this embodiment of the invention, a series of glucose standard solutions of different concentrations were prepared. The standard curve of glucose was determined by the phenol-concentrated sulfuric acid method, and the linear relationship between its concentration and absorbance was investigated. The specific method is as follows: 5.0 mg of anhydrous glucose standard was accurately weighed and diluted to a 50 mL volumetric flask with distilled water. 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the solution were then accurately pipetted into stoppered test tubes and diluted with distilled water to concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg / mL, respectively. 1.0 mL of 5% phenol solution was added and vortexed to mix. 5.0 mL of concentrated sulfuric acid was slowly added along the wall of the tube, and the mixture was shaken well and then boiled in a water bath for 20 min. Using distilled water as a control, the solution was cooled to room temperature, and the absorbance was measured at 490 nm after a full wavelength scan. The standard curve is shown below. Figure 1 As shown, from Figure 1 As can be seen, glucose exhibits good linearity within the concentration range of 0.01-0.06 mg / mL, with the standard equation being y = 9.262x - 0.004, R0. 2 =0.9971.

[0031] Example 1: In Example 1, a series of yam polysaccharide extracts were prepared under different ultrasonic times (4, 6, 8, 10, 12 min). The specific preparation steps are as follows: S1. Preparation of ethanol solution of yam powder: Fresh yam (produced in Jiaozuo City, Henan Province) was washed, air-dried, and then pulverized with a pulverizer. After passing through a 40-mesh sieve, 1.0 g of yam powder was mixed with 30 mL of ethanol to prepare a liquid-to-solid ratio of 30 mL / g yam powder ethanol solution; S2. The above solution was placed in a dual-frequency ultrasonic device (KQ-600DE type CNC ultrasonic cleaner) for ultrasonic-assisted extraction. The ultrasonic conditions were: ultrasonic frequency 40 kHz and 25 kHz, ultrasonic power 435 W (300 W for fixed tank ultrasonic and 135 W for changing probe ultrasonic), and ultrasonic times of 4, 6, 8, 10, and 12 min, respectively; S3. The extract was centrifuged at 5500 rpm for 10 min, filtered, and the series of yam polysaccharide extracts of Example 1 were obtained.

[0032] The extraction rate of yam polysaccharides in Example 1 above was calculated, and the results are as follows: Figure 2 As shown, the extraction rate of yam polysaccharides initially increased and then decreased with increasing ultrasonic time, reaching its maximum at 10 minutes. This indicates that as ultrasonic time increases, yam polysaccharides continuously diffuse into the solvent. However, with excessively long ultrasonic times, the ultrasonic shearing effect degrades the yam polysaccharides, leading to a decrease in the extraction rate. Therefore, the optimal ultrasonic time is 10 minutes.

[0033] Example 2: In Example 2, a series of yam polysaccharide extracts with different liquid-to-solid ratios (10, 20, 30, 40, 50 mL / g) were prepared. The specific preparation steps are as follows: S1. Preparation of ethanol solutions of yam powder: Fresh yam (produced in Jiaozuo City, Henan Province) was washed, air-dried, and then pulverized with a pulverizer. After passing through a 40-mesh sieve, 1.0 g of yam powder was mixed with ethanol to prepare a series of yam powder ethanol solutions with liquid-to-solid ratios of 10, 20, 30, 40, and 50 mL / g; S2. The above solutions were placed in a dual-frequency ultrasonic device (KQ-600DE type CNC ultrasonic cleaner) for ultrasonic-assisted extraction. The ultrasonic conditions were: ultrasonic frequency 40 kHz and 25 kHz, ultrasonic power 435 W (300 W for fixed tank ultrasonic and 135 W for changing probe ultrasonic), and ultrasonic time 10 min; S3. The extract was centrifuged at 5500 rpm for 10 min, filtered, and the series of yam polysaccharide extracts of Example 2 were obtained.

[0034] The extraction rate of yam polysaccharides in Example 2 above was calculated, and the results are as follows: Figure 3 As shown, the extraction rate of yam polysaccharides generally increased and then decreased with the increase of the liquid-to-solid ratio, reaching its highest value at 30 mL / g. This indicates that increasing the liquid-to-solid ratio within a certain range can increase the solid-liquid contact area and facilitate the formation of more uniform ultrasonic cavitation, thus improving the extraction rate of yam polysaccharides. However, if the liquid-to-solid ratio is too high, the ultrasonic energy will be dispersed, leading to a decrease in the extraction rate of yam polysaccharides. Therefore, the optimal liquid-to-solid ratio is 30 mL / g.

[0035] Example 3: In this example, a series of yam polysaccharide extracts were prepared under different ultrasonic powers (345, 390, 435, 480, 525 W). The specific preparation steps are as follows: S1. Preparation of ethanol solution of yam powder: Fresh yam (produced in Jiaozuo City, Henan Province) was washed, air-dried, and then pulverized with a pulverizer. After passing through a 40-mesh sieve, 1.0 g of yam powder was mixed with 30 mL of ethanol to prepare a liquid-to-solid ratio of 30 mL / g yam powder ethanol solution; S2. The above solution was placed in a dual-frequency ultrasonic device (KQ-600D) respectively. Ultrasonic extraction was performed using an E-type CNC ultrasonic cleaner. The ultrasonic conditions were as follows: ultrasonic frequencies of 40kHz and 25kHz, ultrasonic powers of 345, 390, 435, 480, and 525W (300W for fixed tank ultrasonic and 45W, 90W, 135W, 180W, and 225W for changing probe ultrasonic), and ultrasonic time of 10min. S3. The extract was centrifuged at 5500rpm for 10min and filtered to obtain a series of yam polysaccharide extracts of Example 3.

[0036] The extraction rate of yam polysaccharides in Example 3 above was calculated, and the results are as follows: Figure 4As shown, increasing the ultrasonic power initially increases the extraction rate of yam polysaccharides, then decreases, with the highest extraction rate achieved at 435W. This is because increasing the ultrasonic power enhances the cavitation and mechanical effects, which facilitates cell wall disruption and polysaccharide dissolution. However, excessively high ultrasonic power can degrade the polysaccharides to some extent, reducing the extraction rate. Therefore, the optimal ultrasonic power is 435W.

[0037] Example 4: Optimization of dual-frequency ultrasonic extraction process of yam polysaccharide using response surface methodology.

[0038] In this example, Example 4 uses ultrasonic time, liquid-to-solid ratio, and ultrasonic power as independent variables, and polysaccharide extraction rate as the response value. A three-factor, three-level Box-Behnken experiment was designed using Design-Expert 10.0.1 software to prepare a series of yam polysaccharide extracts under different factors (ultrasonic power, liquid-to-solid ratio, ultrasonic time). The specific preparation steps are as follows: S1. Preparation of ethanol solutions of yam powder: Fresh yam (produced in Jiaozuo City, Henan Province) was washed, naturally dried, and then pulverized using a pulverizer. After passing through a 40-mesh sieve, 1.0g of yam powder was mixed with ethanol to prepare yam powder ethanol solutions with different liquid-to-solid ratios (20, 30, 40mL / g); S2. The above solutions were placed in... Ultrasonic extraction was performed using a dual-frequency ultrasonic device (KQ-600DE type CNC ultrasonic cleaner). The ultrasonic conditions were: ultrasonic frequencies of 40kHz and 25kHz, ultrasonic powers of 435, 480, and 525W respectively (300W for fixed tank ultrasonic and 135W, 180W, and 225W for changing probe ultrasonic), and ultrasonic times of 8, 10, and 12 min. S3. The extract was centrifuged at 5500 rpm for 10 min and filtered to obtain a series of yam polysaccharide extracts as described in Example 4.

[0039] The test factors and levels are shown in Table 1, and the test scheme and test results are shown in Table 2. A total of 17 groups were tested, of which 12 groups were three-dimensional factorial points composed of ultrasonic time, liquid-to-material ratio and ultrasonic power independent variables at different levels, and the remaining 5 groups were response surface center repeat experimental points.

[0040] Table 1: Test factors and levels of yam polysaccharides

[0041] Table 2: Response Surface Experiment Design and Test Results

[0042] The data in Table 2 were fitted, and the fitting results are shown in Table 3 below. The model shows a low P-value (<0.0001, highly significant) and a high F-value (78.16), confirming that the quadratic polynomial fitting equation is ideal and that the model has high significance, indicating the reliability of the experimental method. The lack-of-fit term (P=0.9566>0.05) is not significant, indicating a good variance fit and small error, verifying the feasibility of the established regression model. Furthermore, the R-value of this model... 2 The value is 0.9901, R 2 The adj=0.9775 indicates that the model fits the experimental results well, with a small deviation between the actual results and the predicted values; furthermore, the precision (Adeq Precisior) is as high as 25.661, demonstrating the model's strong correlation and the feasibility of the experimental design. Meanwhile, the results show that A, B, C, and A... 2 B 2 C 2 The effects on polysaccharide extraction rate were significant (P < 0.05), with B showing a higher significance than A and C. Therefore, the order of polysaccharide extraction strength was: liquid-to-solid ratio > ultrasonic time > ultrasonic power. The quadratic model fitted in this invention is highly significant and effective.

[0043] The regression equation is as follows: Y=6.41+0.29A+0.47B+0.21C+0.15AB-0.30AC-0.039BC-0.45A 2 -1.04B 2 -1.07C 2 (A: Ultrasonic time, B: Liquid-to-material ratio, C: Ultrasonic power) Table 3: Multiple Regression Fit Analysis

[0044] Note: ns, not significant; **, significant and p < 0.001; *, significant and p < 0.05. (Notes lacking *** need to be modified to correspond with those in the table.) Figure 5 The figures show two-dimensional contour plots (A, C, E) and three-dimensional response surface plots (B, D, F) for dual-frequency ultrasound-assisted extraction of yam polysaccharides. The slope of the response surface plots determines the influence of these two factors on the response values; a larger slope indicates a more significant interaction between the two factors. It can be seen that the contour plot of the BC interaction does not show a clear ellipse shape, but is closer to a circle, indicating that their interaction is not significant, consistent with the results of the analysis of variance. The interaction plots of AB and AC in the figures are nearly elliptical, indicating that their interaction is significant.

[0045] Differentiating the model equations and finding the extreme values, the optimal extraction conditions for yam polysaccharides under the coordination of three factors were determined to be: ultrasonic time 10.70 min, liquid-to-solid ratio 32.51 mL / g, ultrasonic power 439.89 W, and extraction rate 6.521%. Considering actual production factors, the optimal ultrasonic time for the dual-frequency ultrasonic-assisted extraction method of yam polysaccharides described in this invention is 10.70 min, 32.50 mL / g, and 435 W.

[0046] The present invention will be further described below with reference to application examples.

[0047] Application Example 1: Extraction of yam polysaccharides using a dual-frequency ultrasound-assisted extraction method Example 1 of this application uses a dual-frequency ultrasonic-assisted extraction method to extract yam polysaccharides. The specific steps are as follows: S1. Preparation of ethanol solution of yam powder: Wash fresh yam (produced in Jiaozuo City, Henan Province), air dry, and then crush it with a pulverizer. After passing through a 40-mesh sieve, take 1.0g of yam powder and mix it with 32.5mL of ethanol to prepare a liquid-to-solid ratio of 32.5mL / g yam powder ethanol solution; S2. Place the above solution in a dual-frequency ultrasonic device (KQ-600DE type CNC ultrasonic cleaner) for ultrasonic-assisted extraction. The ultrasonic conditions are: ultrasonic frequency 40kHz and 25kHz, ultrasonic power 435W (300W for fixed tank ultrasonic and 135W for changing probe ultrasonic), and ultrasonic time 10.7min; S3. Centrifuge the extract at 5500rpm for 10min, filter, and obtain the yam polysaccharide extract of Example 1.

[0048] Comparative Example 1: Ultrasonic-assisted cellulase extraction of yam polysaccharides Comparative Example 1 uses ultrasound-assisted cellulase extraction to extract yam polysaccharides. The specific steps are as follows: the optimal extraction process is a material-to-liquid ratio of 1:20 (g / mL), an ultrasound time of 20 min, an ultrasound power of 450 W, and an enzyme addition of 2%.

[0049] Comparative Example 2: Extraction of yam polysaccharides using ultra-high pressure-lactic acid bacteria fermentation synergistic extraction method Comparative Example 2 uses an ultra-high pressure-lactic acid bacteria fermentation synergistic extraction method to extract yam polysaccharides. The specific steps are as follows: inoculum concentration 5%, fermentation time 2 days, material-liquid ratio 1:5 (m / V), pressure 500 MPa, and holding time 10 min.

[0050] Comparative Example 3: Extraction of yam polysaccharides by hot water extraction Comparative Example 3 uses hot water extraction to extract yam polysaccharides. The specific steps are as follows: after mixing at a ratio of 1:10 (w / v), it was extracted at 80°C for 2 hours.

[0051] Comparative Example 4: Extraction of yam polysaccharides using single-frequency ultrasonic extraction method Comparative Example 4 uses single-frequency ultrasonic extraction to extract yam polysaccharides. The specific steps are as follows: the ratio of material to liquid is 1:25. Ultrasonic extraction is performed at 70°C for 90 minutes.

[0052] Comparative Example 5: Extraction of yam polysaccharides by deep eutectic solvent extraction Comparative Example 5 uses deep eutectic solvent extraction to extract yam polysaccharides. The specific steps are as follows: DES composed of choline chloride and 1,4-butanediol can effectively extract polysaccharides. The ideal extraction conditions are 32.89% water content, 94.00℃ extraction temperature, and 44.74 min extraction time.

[0053] Comparative Example 6: Extraction of yam polysaccharides using cellulase-assisted extraction method Comparative Example 6 used a cellulase-assisted extraction method to extract yam polysaccharides. The specific steps were as follows: Cellulase was dissolved in distilled water (1:1, g / mL). 10 g of Chinese yam powder was mixed with 150 mL of distilled water, and then stirred at room temperature for 30 minutes. Extraction was then carried out in a 50°C water bath for a fixed time (40–120 minutes). The reaction was stopped by heating at 90°C for 10 minutes to inactivate the enzyme.

[0054] Comparative Example 7: Extraction of yam polysaccharides by ultrafiltration-assisted extraction Comparative Example 7 used ultrafiltration-assisted extraction to extract yam polysaccharides. The specific steps are as follows: pH 6.5, temperature 20℃, pressure 0.03 MPa. (Complete steps are required for the above test methods.) The extraction rates of Application Example 1 and Comparative Examples 1-7 were tested, and their process efficiency was analyzed. The results are shown in Table 4 below: Table 4: Analysis of Extraction Rate and Efficiency of Yam Polysaccharide Extraction Process

[0055] The above results demonstrate the significant advantages of the dual-frequency ultrasound-assisted extraction method of this invention: In terms of process efficiency, the method of this invention, through the synergistic effect of dual frequencies at 435W ultrasonic power, can complete the extraction process in just 10 minutes, making it the shortest process besides ultrafiltration-assisted extraction. This is far faster than hot water extraction (requiring 2 hours), conventional ultrasonic extraction (90 minutes), and deep eutectic solvent extraction (requiring 44.74 minutes), greatly improving production turnover. Meanwhile, its extraction rate of 6.30%, while significantly higher than hot water extraction (0.6%) and conventional ultrasonic extraction (2.7%), reflects the superiority of dual-frequency technology in disrupting cell structure and promoting solute diffusion. Furthermore, compared to complex extraction technologies requiring multiple steps or the addition of exogenous reagents (such as ultrasound-assisted enzymatic methods and ultrafiltration-assisted extraction), the process flow of this invention is significantly simplified. It eliminates the need for biological enzymes, chemical reagents, or additional separation equipment; efficient extraction can be achieved simply through parameter optimization, reducing process complexity and control difficulty, and facilitating stable implementation and cost control in industrial production. In summary, the dual-frequency ultrasound-assisted extraction method provided by this invention is an efficient, rapid, and easy-to-operate process for extracting yam polysaccharides. It is particularly suitable for industrial production scenarios with strict requirements on extraction time, and at the same time provides a reliable technical basis for subsequent process compounding and optimization.

[0056] The present invention also conducted an in vitro antioxidant test on the yam polysaccharide extract prepared in the above application example 1. The test method is as follows: 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging test: Prepare sample solutions of different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mg / mL). Take 2 mL of each solution into a 5 mL test tube, add 2 mL of DPPH solution (0.1 mmol / L), mix well, and react in the dark for 30 min. Measure the absorbance at 517 nm using a UV-Vis spectrophotometer. Using ascorbic acid (VC) as a positive control, the DPPH free radical scavenging rate is calculated using the following formula:

[0057] A1 is the absorbance of the sample after mixing with DPPH, A2 is the absorbance of the sample solution, and A0 is the absorbance of the DPPH solution.

[0058] 2,2'-Aza-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging test: Mix 7.4 mmol / L ABTS solution with 2.5 mmol / L potassium persulfate solution at a 1:1 ratio, and incubate in the dark for 12-16 hours to obtain ABTS• +Dilute the solution to a absorbance of 0.7 ± 0.02 at 734 nm for later use. Prepare sample solutions of different concentrations (1.0, 2.0, 3.0, 4.0, 0.5 mg / mL), take 1 mL in a 5 mL test tube, and add 3 mL of ABTS• + Mix thoroughly. After incubating at room temperature in the dark for 6 minutes, measure the absorbance at 734 nm using a UV-Vis spectrophotometer. Using VC as a positive control, the ABTS free radical scavenging rate is calculated using the following formula:

[0059] A1 is the sample and ABTS• + The absorbance after mixing, A2 is the absorbance of the sample solution, and A0 is the absorbance of ABTS• + The absorbance of the solution.

[0060] Hydroxyl radical scavenging test: Accurately weigh 0.0829 g of salicylic acid and dilute to 100 mL in a volumetric flask with ethanol to prepare a 6 mmol / L salicylic acid-ethanol solution. Accurately measure 68 μL of 30% hydrogen peroxide solution and dilute to 100 mL in a volumetric flask with water to prepare a 6 mmol / L hydrogen peroxide solution. Accurately weigh 0.1668 g of ferrous sulfate heptahydrate and dilute to 100 mL in a volumetric flask with water to prepare a 6 mmol / L ferrous sulfate solution. In a 10 mL test tube, add 1 mL each of the salicylic acid-ethanol solution, FeSO4 solution, sample solutions of different concentrations (1.0, 2.0, 3.0, 4.0, 5.0 mg / mL), and 6 mmol / L H2O2. Mix thoroughly and react at room temperature for 30 min. Then, allow to stand at room temperature in the dark for 25 min. Measure the absorbance A1 at 510 nm using a UV-Vis spectrophotometer. Using VC as a positive control, the hydroxyl radical scavenging rate is calculated using the following formula:

[0061] A1 is the absorbance of the sample after mixing with •OH, A2 is the absorbance of the sample solution, and A0 is the absorbance of the •OH solution.

[0062] Iron reducing power test: Different concentrations of sample solutions (5, 6, 7, 8, 9 mg / mL) were prepared using the potassium ferricyanide colorimetric method. 1 mL of each solution was placed in a 10 mL test tube, and 1 mL of 1% potassium ferricyanide solution and 1 mL of 0.2 mol / L phosphate buffer were added. The mixture was placed in a 50℃ water bath for 20 min, cooled under running water, and immediately 1 mL of 10% trichloroacetic acid was added. The mixture was centrifuged (3000 r / min) for 10 min. 2 mL of the supernatant was taken, and 2 mL of deionized water and 0.4 mL of 0.1% FeCl3 solution were added. After mixing thoroughly and standing for 10 min, the absorbance was measured at 700 nm using a UV-Vis spectrophotometer. Using vitamin C as a positive control, the iron reducing power was calculated using the following formula:

[0063] A1 is the absorbance of the sample solution after mixing with FeCl3, and A0 is the absorbance of the sample solution.

[0064] Figure 6 The figure shows the DPPH free radical scavenging test results of the yam polysaccharide prepared by the method of this invention. As can be seen from the figure, DPPH is a stable purple free radical with strong absorption at 517 nm. When an antioxidant provides hydrogen atoms to combine with DPPH, DPPH is reduced to a yellow non-free radical form. Within the range of 0.5-2.5 mg / mL, yam polysaccharide exhibits strong DPPH free radical scavenging ability, and the DPPH free radical scavenging rate of the sample solution increases significantly with increasing concentration, showing a concentration-dependent effect. At a sample concentration of 2 mg / mL, the scavenging rate reaches approximately 80%, indicating good antioxidant activity of yam polysaccharide.

[0065] Figure 7 The graph shows the ABTS free radical scavenging test results of the yam polysaccharide prepared by the method of this invention. As can be seen from the graph, ABTS is oxidized by the oxidant potassium persulfate to produce blue-green ABTS•. + It has absorption at 734 nm, and the antioxidant can reduce ABTS• + Within the range of 1.0-5.0 mg / mL, yam polysaccharide exhibits strong scavenging ability against ABTS free radicals, and this scavenging ability is concentration-dependent, with the scavenging rate increasing with increasing sample concentration. At a sample concentration of 5 mg / mL, the scavenging rate reached 90%.

[0066] Figure 8The figure shows the hydroxyl radical scavenging test results of the yam polysaccharide prepared by the method of this invention. As can be seen from the figure, the experiment generates highly reactive •OH through the Fenton reaction, and the antioxidant can competitively scavenge •OH. The chromogenic agent salicylic acid can capture unremoved •OH to generate a colored product, which is detected at 510 nm. Within the experimental concentration range, yam polysaccharide (1.0-5.0 mg / mL) has a hydroxyl radical scavenging effect, and its scavenging ability is concentration-dependent, increasing with increasing concentration. Compared with ABTS, it has a stronger hydroxyl radical scavenging rate; at a sample solution concentration of 3 mg / mL, the hydroxyl radical scavenging rate is 90%.

[0067] Figure 9 The graph shows the iron reducing power test results of the yam polysaccharide prepared by the method of this invention. As can be seen from the graph, the antioxidant can reduce Fe... 3+ Reduced to Fe 2+ The absorbance was measured at 593 nm. For samples with concentrations ranging from 5.0 to 9.0 mg / mL, the iron reducing power increased with increasing yam polysaccharide concentration, indicating that yam polysaccharide possesses a certain reducing ability.

[0068] It is understood that the above specific embodiments are all further illustrations of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, all other modifications and refinements obtained without creative effort are within the scope of protection of the present invention.

Claims

1. A method for dual-frequency ultrasound-assisted extraction of yam polysaccharides, characterized in that, Includes the following steps: S1. Prepare an ethanol solution of yam powder; S2. Place the above solution in a dual-frequency ultrasonic device for ultrasonic-assisted extraction. The ultrasonic conditions include: ultrasonic frequency of 40kHz and 25kHz, ultrasonic power of 345-525W, and ultrasonic time of 4-12min. S3. After filtration, yam polysaccharide extract is obtained.

2. The method for dual-frequency ultrasound-assisted extraction of yam polysaccharides according to claim 1, characterized in that, In step S1, the liquid-to-solid ratio of yam powder to ethanol is 10-50 mL / g.

3. The dual-frequency ultrasound-assisted extraction method for yam polysaccharides according to claim 2, characterized in that, The liquid-to-solid ratio is 25-40 mL / g.

4. The dual-frequency ultrasound-assisted extraction method for yam polysaccharides according to claim 3, characterized in that, The liquid-to-solid ratio is 32.5 mL / g.

5. The method for dual-frequency ultrasound-assisted extraction of yam polysaccharides according to claim 1, characterized in that, In step S2, the ultrasonic power is 390-480W.

6. The method for dual-frequency ultrasound-assisted extraction of yam polysaccharides according to claim 5, characterized in that, The ultrasonic power is 435W.

7. The method for dual-frequency ultrasound-assisted extraction of yam polysaccharides according to claim 1, characterized in that, In step S2, the ultrasound time is 8-12 minutes.

8. The method for dual-frequency ultrasound-assisted extraction of yam polysaccharides according to claim 7, characterized in that, The ultrasound time was 10.7 min.

9. A yam polysaccharide extracted by the dual-frequency ultrasound-assisted extraction method according to any one of claims 1-8.

10. The use of the yam polysaccharide according to claim 9 in the preparation of antioxidant functional foods, pharmaceuticals and cosmetics.

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