Process for extracting anredera cordifolia bulbil polysaccharide by acid method and application thereof

Abstract

A process for extracting anredera cordifolia bulbil polysaccharide by an acid method comprises the following steps: S1, pretreatment: washing, drying, crushing and sieving anredera cordifolia bulbil to obtain anredera cordifolia bulbil powder; s2, acid liquid extraction: mixing the anredera cordifolia bulbil powder with a diluted hydrochloric acid solution, and performing water bath heating extraction under the conditions that the extraction temperature is 70-85 DEG C, the pH value is 4.5-5.5, the heating time is 50-70 minutes and the material-liquid ratio is (1: 35)-(1: 45) g / mL to obtain an extracting solution; and S3, separation and purification: cooling the extracting solution, centrifuging, taking supernate, adding ethanol, carrying out alcohol precipitation, centrifuging, collecting precipitate, and carrying out deproteinization, dialysis and freeze drying to obtain the anredera cordifolia bulbil polysaccharide. The method is obviously higher than existing extraction methods such as a traditional hot water extraction method, and the extraction efficiency of the anredera cordifolia bulbil polysaccharide is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide extraction technology, specifically to an acid extraction process for polysaccharides from Malabar spinach bulbils and its application. Background Technology

[0002] Malabar spinach, also known as vine ginseng, is a perennial succulent vine belonging to the genus Malabar spinach in the family Basellaceae. It can grow to several meters long and has a large yield and is easy to obtain. Malabar spinach bulbils are the tuberous buds on the Malabar spinach vine. Malabar spinach bulbils have a high polysaccharide content, and long-term consumption can promote metabolism, delay cell aging, and have beauty and health benefits.

[0003] Polysaccharides, as important biomolecules in organisms, are natural polymers linked together by glycosidic bonds and are essential for maintaining life activities. There are many methods for extracting plant polysaccharides, mainly including microwave-assisted extraction, ultrasonic extraction, hot water extraction, and enzymatic extraction. Numerous studies have shown that different extraction methods have a significant impact on the extraction rate, chemical composition, and antioxidant activity of polysaccharides.

[0004] Acid extraction, developed from hot water extraction, utilizes the action of acids (such as hydrochloric acid) to cause plant cells to absorb water, swell, and rupture, thereby releasing free polysaccharides and improving extraction efficiency. Compared with other extraction methods, acid extraction has advantages such as simple equipment requirements, convenient operation, and lower cost. Furthermore, plant polysaccharides extracted by acid extraction often possess high physiological activity and can even replace the functions of some water-extracted plant polysaccharides. However, the control of acid concentration and extraction conditions is crucial in acid extraction. Excessively high acid concentrations can cause the breakage of glycosidic bonds in polysaccharides, leading to a decrease in extraction rate and physiological activity. Therefore, lower concentrations of acid are required for extraction. Currently, a systematic optimization scheme for acid extraction of Malabar spinach bulbil polysaccharides has not yet been developed. The influence of key parameters such as extraction temperature, pH value, heating time, and solid-liquid ratio on the extraction rate is unclear, hindering the efficient extraction and large-scale development and utilization of Malabar spinach bulbil polysaccharides. Summary of the Invention

[0005] The present invention aims to provide an acid extraction process for polysaccharides from Malabar spinach bulbils and its application, which utilizes the effect of dilute hydrochloric acid on Malabar spinach bulbil cells to achieve efficient extraction of polysaccharides from Malabar spinach bulbils.

[0006] To achieve the above objectives, the following technical solution is provided: A process for acid extraction of polysaccharides from Malabar spinach bulbils includes the following steps: S1, Pretreatment: Malabar spinach bulbils are washed, dried, crushed, and sieved to obtain Malabar spinach bulbil powder; S2, Acid extraction: Malabar spinach bulbil powder is mixed with dilute hydrochloric acid solution and extracted by water bath heating at an extraction temperature of 70-85℃, a pH of 4.5-5.5, a heating time of 50-70 min, and a material-to-liquid ratio of 1:35-1:45 g / mL to obtain an extract; S3, Separation and purification: The extract is cooled and centrifuged, the supernatant is collected, ethanol is added for alcohol precipitation, the precipitate is collected by centrifugation, and after protein removal, dialysis, and freeze-drying, Malabar spinach bulbil polysaccharides are obtained.

[0007] As a preferred embodiment, the extraction temperature is 78.4℃, the pH value is 4.9, the heating time is 60 min, and the material-to-liquid ratio is 1:41 g / mL.

[0008] As a preferred embodiment, the concentration of the dilute hydrochloric acid solution is 0.05–0.15 mol / L, and the stirring rate during water bath heating is 100–200 r / min.

[0009] As a preferred method, the centrifugation speed is 3500-4500 r / min and the centrifugation time is 10-20 min when collecting the sediment by centrifugation.

[0010] As a preferred method, the final ethanol concentration during alcohol precipitation is 85-95%, and the precipitation time is 20-28 hours.

[0011] As a preferred method, when removing protein, chloroform-n-butanol mixed in a 4:1 ratio is combined with polysaccharide extract in a 1:5 ratio, and the upper aqueous phase is collected after centrifugation.

[0012] Application of a Malabar spinach bulbil polysaccharide in the preparation of antioxidants.

[0013] Application of a Malabar spinach bulbil polysaccharide in the preparation of health products or food additives.

[0014] Working principle and beneficial effects of the present invention: The optimal extraction parameters for Malabar spinach bulbil polysaccharide using the acid method in this application are: extraction temperature 78.4℃, pH 4.9, heating time 60 min, and solid-liquid ratio 1:41 g / mL. Under these conditions, the extraction rate of Malabar spinach bulbil polysaccharide can reach 20.54 mg / g, which is significantly higher than existing extraction methods such as traditional hot water extraction, effectively improving the extraction efficiency of Malabar spinach bulbil polysaccharide. Attached Figure Description

[0015] Figure 1 The effect of extraction temperature on the polysaccharide content of Malabar spinach bulbils; Figure 2 The effect of pH on the polysaccharide content of Malabar spinach bulbils; Figure 3 The effect of water bath time on polysaccharide content in Malabar spinach bulbils; Figure 4 The effect of the material-to-liquid ratio on the polysaccharide content of Malabar spinach bulbils; Figure 5 The scavenging rate of DPPH free radicals by Malabar spinach bulbil polysaccharide; Figure 6 The scavenging rate of ABTS free radicals by Malabar spinach bulbil polysaccharide; Figure 7 Scanning electron microscopy image of Malabar spinach bulbils polysaccharide; Figure 8 Infrared spectrum of Malabar spinach bulbils polysaccharide; Figure 9 X-ray diffraction pattern of Malabar spinach bulbils polysaccharide. Detailed Implementation

[0016] The following detailed description illustrates the specific implementation method: Example: An acid extraction process for polysaccharides from Malabar spinach bulbils. After washing, Malabar spinach bulbils are dried at 80°C for 72 hours and then pulverized through a 200-mesh sieve. 0.1000 g of powder is weighed, and an acid solution of 1:40 (g / mL) pH=4 is added. The mixture is heated in a water bath at 70°C for 90 minutes, centrifuged at 4000 r / min for 15 minutes, and the supernatant is collected. Ethanol is added to a concentration >90%, and the mixture is precipitated for 24 hours. The precipitate is then collected by centrifugation, and the precipitate is further subjected to protein removal, dialysis, and freeze-drying to obtain the freeze-dried polysaccharide sample.

[0017] (1) Single-factor experiment: Three parameters were fixed, and the effects of extraction temperature of 50-90℃, pH 2-6, heating time of 30-150min and material-liquid ratio of 1:20-1:60 (g / mL) on polysaccharide yield were investigated.

[0018] (2) Response surface design: Based on the results of the single factor, the polysaccharide yield was used as the response value. The Box-Behnken model was used to design a 4-factor (extraction temperature, pH, heating time, material-liquid ratio) 3-level experiment, with the level codes being -1, 0, and 1 (corresponding to temperatures of 60 / 80 / 100℃, pH of 4 / 5 / 6, time of 30 / 60 / 90 min, and material-liquid ratio of 1:20 / 1:40 / 1:60).

[0019] (3) Antioxidant assay: DPPH / ABTS free radical scavenging assay: The sample and corresponding reagents were mixed and reacted for 30 min. The absorbance was measured, and the scavenging rate and IC50 were calculated. 50 ; (4) Structural characterization: Monosaccharide composition: Polysaccharides were hydrolyzed by TFA, washed with methanol, and then measured using a chromatographic column; Molecular weight: Samples were dissolved in 0.1M NaNO3 or DMSO, filtered, and then detected; Scanning electron microscopy: Samples were sieved and sputtered with gold before observation; Infrared spectroscopy: Polysaccharides were mixed with KBr and pressed into pellets for measurement; X-ray diffraction: Samples were ground and sieved, and X-rays were observed at θ / 2θ5. ° -40 ° Range scan.

[0020] (5) Polysaccharide determination: Prepare 5% phenol solution and 4 mg / mL glucose standard solution, prepare standard solutions of different concentrations, add phenol and concentrated sulfuric acid to react and measure absorbance value, and plot standard curve; measure absorbance value of sample in the same way, substitute into curve to calculate concentration.

[0021] The reference results are as follows: (1) Single-factor experiment reference Figures 1-4 , Extraction temperature: Polysaccharide yield increases at 50-80℃, reaching 20.32 mg / g at 80℃ (P<0.05). At 90℃, there is no yield increase and energy consumption is high; 80℃ is preferable. pH: Yield increases at pH 2-5, reaching its highest at pH 5 (P<0.05). Yield decreases at pH > 5 due to insufficient cell wall disruption caused by weakened acidity; pH ​​5 is preferable. Heating time: Yield increases at 30-60 min, reaching 15.64 mg / g at 60℃. P<0.05 Impurities dissolve within 60 minutes, hindering polysaccharide dissolution; therefore, a time of around 60 minutes is recommended. For the material-to-liquid ratio: 1:20-1:40 increases yield, reaching 16.02 mg / g at 1:40 (P<0.05); a ratio >1:40 decreases polysaccharide concentration; therefore, a ratio of around 1:40 is recommended.

[0022] (2) Based on the single-factor experiment, the regression equation is obtained: Y=-150.1352+2.0538X1+27.5714X2+0.3988X3+0.5190X4+0.0270X1X2-0. 00058X1X3-0.00066X1X4-0.0049X2X3+0.0049X2X4-0.0007X3X4-0.0136X1 2 -3.0368X2 2 -0.0025X3 2 -0.0055X4, the model is highly significant (F=25.73). P<0.0001 The lack-of-fit term was not significant (P=0.1989), R 2 =0.9626, R 2 Adj=0.9252, predictable yield; influence of factors: X1 (temperature) > X2 (pH) > X4 (solid-to-material ratio) > X3 (time).

[0023] The predicted optimal process is 78.4℃, pH 4.90, 60 min, and 1:41 (g / mL), with a theoretical yield of 20.652 mg / g. The verified yield is 20.54 mg / g, which is close to the theoretical value, indicating that the model is reliable.

[0024] (3) From Figure 5 The results show that the scavenging rate of DPPH free radicals by Malabar spinach bulbil polysaccharide increases with increasing concentration (0 mg / mL to 6 mg / mL). At a concentration of 3 mg / mL, the scavenging rate reaches 71.36%. Nonlinear curve regression analysis of the data yields the equation y = -0.032x. 2 +0.3271x+0.0028, R 2 =0.9977, and the half-maximal inhibitory concentration (IC50) of Malabar spinach bulbil polysaccharide for DPPH free radical scavenging was calculated. 50 The concentration was 1.86 mg / mL.

[0025] (4) By Figure 6 The scavenging rate of ABTS free radicals by Malabar spinach bulbil polysaccharides showed that, within the concentration range of 0.25 mg / mL to 8 mg / mL, the ability to scavenge ABTS free radicals gradually increased with increasing polysaccharide content. When the polysaccharide concentration was 6 mg / mL, the scavenging rate was 59.68%, and when the concentration reached 8 mg / mL, the scavenging rate was 68.68%. Processing the data in the figure yielded the curve equation: y = -0.0066x 2 +0.1357x+0.0234, R 2 =0.9994, the IC50 of Malabar spinach bulbil polysaccharide for scavenging ABTS free radicals was calculated. 50 The value was 4.49 mg / mL.

[0026] (5) Determine the monosaccharide composition and polysaccharide molecular weight distribution, refer to Table 1: Table 1. Monosaccharide percentage of Malabar spinach bulbils polysaccharides

[0027] The main components of Malabar spinach bulbil polysaccharide are galactose and glucose, accounting for 36.47% and 31.10% respectively, followed by arabinose (24.51%) and rhamnose (5.38%). A peak appears between ribose and galacturonic acid, but no corresponding monosaccharide control was found, suggesting that it may be a metabolite with a similar molecular weight to the monosaccharide.

[0028] (6) Scanning electron microscopy analysis like Figure 7 These are scanning electron microscope images of Malabar spinach bulbils polysaccharide at magnifications of 500, 1K, 2K, and 10K. As shown in the image below, the Malabar spinach bulbils polysaccharide is distributed in blocks of varying sizes, with some structures exhibiting a grid-like structure. The surface is relatively rough and has varying layers.

[0029] (7) Infrared spectroscopy analysis Figure 8 The image shows the infrared spectrum of Malabar spinach bulbil polysaccharide. As can be seen, a strong and broad smooth absorption peak appears near 3424 cm⁻¹, which is caused by the stretching vibration of the –OH group. The absorption peak near 2930 cm⁻¹ is caused by the C–H stretching vibration. Both of these absorption peaks are typical of polysaccharides. (1639 cm⁻¹) -1 The absorption peak nearby is likely the absorption peak of C=O (carbonyl group), at 1410 cm⁻¹. -1 The absorption peak at 1080° is caused by the C–H angle vibration, while the absorption peak at 1080° is caused by the C–H angle vibration. -1 The presence of an absorption peak indicates that Malabar spinach bulbils polysaccharide has a pyran ring structure.

[0030] (8) X-ray diffraction analysis X-ray diffraction (XRD) analysis revealed the crystal structure information of polysaccharide residues. For example... Figure 9 The image shows the crystal morphology of Malabar spinach bulbil polysaccharide, which exhibits a peak at around 19.88° and no obvious peaks elsewhere. Therefore, it can be determined that Malabar spinach bulbil polysaccharide is an amorphous structure containing a small number of tiny crystals.

[0031] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention, and these should also be considered within the scope of protection of this invention. These modifications and improvements will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An acid extraction process for polysaccharides from Malabar spinach bulbils, characterized in that, Includes the following steps: S1. Pretreatment: Wash, dry, crush and sieve the Malabar spinach bulbils to obtain Malabar spinach bulbil powder; S2. Acid extraction: Malabar spinach bulbil powder is mixed with dilute hydrochloric acid solution and extracted by water bath heating at an extraction temperature of 70-85℃, pH value of 4.5-5.5, heating time of 50-70 min, and material-to-liquid ratio of 1:35-1:45 g / mL to obtain the extract. S3. Separation and purification: After cooling the extract, centrifuge it, add ethanol to the supernatant for alcohol precipitation, centrifuge to collect the precipitate, remove protein, dialyze, and freeze dry to obtain Malabar spinach bulb polysaccharide.

2. The process according to claim 1, characterized in that, The extraction temperature was 78.4℃, the pH value was 4.9, the heating time was 60 min, and the material-to-liquid ratio was 1:41 g / mL.

3. The process according to claim 2, characterized in that, The concentration of the dilute hydrochloric acid solution is 0.05–0.15 mol / L, and the stirring rate during water bath heating is 100–200 r / min.

4. The process according to claim 3, characterized in that, When collecting the precipitate by centrifugation, the centrifugation speed is 3500-4500 r / min and the centrifugation time is 10-20 min.

5. The process according to claim 4, characterized in that, The final ethanol concentration during alcohol precipitation is 85-95%, and the precipitation time is 20-28 hours.

6. The process according to claim 5, characterized in that, When removing protein, mix chloroform-n-butanol in a 4:1 ratio with the polysaccharide extract in a 1:5 ratio, and take the upper aqueous phase after centrifugation.

7. The use of Malabar spinach bulbil polysaccharide according to any one of claims 1 to 5 in the preparation of antioxidants.

8. The use of Malabar spinach bulbil polysaccharide according to any one of claims 1 to 5 in the preparation of health products or food additives.

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