A method for extracting sugarcane polyphenols

Abstract

This invention belongs to the field of natural active substance extraction technology and provides a method for extracting sugarcane polyphenols, including the following steps: fresh sugarcane is crushed into pulp and then freeze-dried to obtain freeze-dried sugarcane pulp, which is then pretreated using a pulsed electric field, followed by ultrasonic-assisted extraction to obtain an extract; after removing the organic solvent from the extract, it is concentrated and freeze-dried to obtain a sugarcane polyphenol extract. This method uses freeze-drying pretreatment, which improves the extraction rate and content of sugarcane polyphenols and avoids the destruction of sugarcane polyphenols by high-temperature treatment. The combined pulsed-ultrasonic synergistic extraction reduces the time and temperature of single ultrasonic extraction, protects the bioactivity of polyphenols, reduces energy consumption, and saves costs, making it a highly efficient and green extraction method.

Description

Technical Field

[0001] This invention relates to the field of natural active substance extraction technology, and more specifically, to a method for extracting sugarcane polyphenols. Background Technology

[0002] Sugarcane polyphenols possess antioxidant stress-reducing properties, showing promising therapeutic effects on a range of diseases caused by oxidative stress, including aging, diabetes, inflammation, and cancer. Studies have demonstrated that the bioactivities of sugarcane polyphenols include antioxidant, anti-inflammatory, anticancer, and antibacterial properties. It is a natural component with significant nutritional and medicinal value, possessing immense development potential and a promising future.

[0003] Currently, the main methods for extracting sugarcane polyphenols include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, and bio-enzymatic extraction. Solvent extraction, as a classic method, has high requirements for extraction processes and solvents; microwave-assisted extraction requires heating, increasing fuel costs; prolonged ultrasound extraction generates thermal effects, affecting the structure and bioactivity of heat-sensitive components; and bio-enzymatic extraction has relatively high economic costs.

[0004] Among the methods described above, ultrasonic extraction is a non-thermal extraction technology, possessing advantages such as being green and safe, having short processing time, and low energy consumption, and thus showing broad application prospects. The acoustic energy generated by ultrasound can form a large number of cavitation bubbles. As the bubbles continuously expand and grow, the internal pressure eventually exceeds the surface tension, causing the bubbles to disintegrate. At this point, a huge shear force is generated within the cavitation region, causing cell damage, while temperature and pressure also rise to a certain extent. However, prolonged ultrasonic treatment will produce a thermal effect, which will affect the structure and biological activity of heat-sensitive components, especially polyphenols.

[0005] Therefore, there is an urgent need to develop a green and efficient method for extracting sugarcane polyphenols, which can improve the extraction rate and protect biological activity, while also reducing energy consumption and saving costs. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of at least one of the above-mentioned prior art and provide a sugarcane polyphenol extraction method to solve the problem of heat generation from ultrasonic treatment destroying the bioactivity of polyphenols, thereby achieving the effect of both improving the extraction rate and protecting the bioactivity of polyphenols.

[0007] The technical solution adopted by this invention is to provide a method for extracting sugarcane polyphenols, comprising the following steps:

[0008] S1: Fresh sugarcane is crushed into residue and then freeze-dried at low temperature to obtain freeze-dried sugarcane residue;

[0009] S2: Mix freeze-dried sugarcane bagasse with ethanol and pre-treat with a pulsed electric field;

[0010] S3: The freeze-dried sugarcane bagasse pretreated with pulsed electric field is ultrasonically extracted to obtain an extract;

[0011] S4: Centrifuge the extract, filter it, collect the filtrate and concentrate it, freeze-dry the concentrate to obtain sugarcane polyphenol extract.

[0012] Furthermore, the pulse processing conditions in step S2 are: pulse intensity of 1–5 KV / cm, pulse width of 8–12 μs, and pulse number of 10–100.

[0013] Furthermore, the conditions for ultrasonic treatment in step S3 are: temperature 30–60°C, power 40–200W, and duration 30–90 min.

[0014] Furthermore, the temperature for low-temperature freeze drying in step S1 is -100 to -70°C.

[0015] Furthermore, the volume of the crushed bagasse in step S1 does not exceed 1 mm. 3 .

[0016] Furthermore, in step S2, the volume ratio of sugarcane bagasse to ethanol is 1:(10-50).

[0017] Furthermore, the centrifugation conditions in step S4 are: 2000–5000g, 5–10min.

[0018] Furthermore, the concentration temperature in step S4 is 40–45°C.

[0019] Step S1 of this scheme uses vacuum freeze-drying technology, which can remove moisture from the sample at low temperature. Compared with traditional heat treatment, it can protect the structure of heat-sensitive polyphenols, and at the same time, it can destroy the cell wall through the process of water vaporization, thereby enhancing the permeability of the cell membrane.

[0020] Step S2 employs pulsed electric field technology. By placing the raw material between two electrodes and applying a pulsed electric field, the structure of cellulose and hemicellulose is disrupted, leading to a rapid and significant increase in cell membrane conductivity and permeability. This causes irreversible electroporation of the sugarcane cell walls and membranes, resulting in cell inactivation and promoting the dissolution of polyphenol-containing cytoplasm. Step S3 uses ultrasonic treatment. Ultrasonic waves induce cavitation in the sugarcane bagasse through continuous mechanical vibration, promoting the dissolution of sugarcane cytoplasm along the electroporation channels formed by the pulsed electric field technology. This pulsed electric field pretreatment step improves polyphenol extraction efficiency, reduces ultrasonic extraction time, and avoids the structural damage and reduced activity of polyphenols caused by excessively long ultrasonic treatment times.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The sugarcane polyphenol extraction method provided by this invention uses low-temperature freeze-drying pretreatment of bagasse. Compared with traditional hot drying pretreatment, this method not only avoids the impact of high temperature on the polyphenol structure and bioactivity, but also, the sublimation of water to a certain extent destroys the plant cell wall and cell membrane, which is more conducive to the dissolution of polyphenols and improves extraction efficiency. Furthermore, the use of pulsed electric field combined with ultrasonic treatment further reduces the temperature and time of ultrasonic treatment, enabling the extraction of high-content polyphenols at low temperature and in a short time, thus avoiding the thermal effects of long-term ultrasonic extraction on heat-sensitive components. Attached Figure Description

[0023] Figure 1 Scanning electron microscope images of the surface morphology of sugarcane after different treatments. Figure 1 a fresh sugarcane, Figure 1 b. Sugarcane subjected to freeze-thaw treatment Figure 1 c. Freeze-dried sugarcane.

[0024] Figure 2 These are scanning electron microscope images of the surface morphology of sugarcane after extraction using different methods. Figure 2 a. Ultrasound, Figure 2 b pulse, Figure 2 c. Microwave Figure 2 d-pulse ultrasound. Detailed Implementation

[0025] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the invention. To better illustrate the following embodiments, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions; it is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0026] Example 1: Comparison Experiment of Sugarcane Conductivity under Different Pretreatments

[0027] To achieve the best freeze-drying pretreatment effect, the electrical conductivity of sugarcane after three pretreatments—room temperature, freeze-drying, and freeze-thaw—was compared. Higher electrical conductivity indicates the greatest degree of damage to sugarcane cells caused by that pretreatment. The surface morphology of sugarcane bagasse after different pretreatments was also compared under a scanning electron microscope. The experimental steps are as follows:

[0028] Purchase fresh sugarcane and crush it into pieces smaller than 1mm. 3 Sugarcane bagasse was freeze-dried at -80℃ to obtain freeze-dried sugarcane bagasse, which was then stored in a desiccator. 1g of freeze-dried sugarcane bagasse was taken, added to 30ml of deionized water, and after standing for 30 minutes, its conductivity was measured using a conductivity meter. The conductivity was measured again after 24 hours, and the difference between the two measurements was calculated and recorded as the freeze-dried sugarcane conductivity.

[0029] Purchase fresh sugarcane and crush it into pieces smaller than 1mm. 3Take 1g of fresh sugarcane bagasse, add 30ml of deionized water, let it stand for 30 minutes, and then measure its conductivity using a conductivity meter. Measure the conductivity again after 24 hours and calculate the difference between the two measurements; this difference is recorded as the fresh sugarcane conductivity.

[0030] Purchase fresh sugarcane and crush it into pieces smaller than 1mm. 3 Sugarcane bagasse was frozen at -20°C for 12 hours, and then left at room temperature for 12 hours to obtain freeze-thawed sugarcane bagasse. 1g of freeze-thawed sugarcane bagasse was taken, added to 30ml of deionized water, and after standing for 30 minutes, its conductivity was measured using a conductivity meter. The conductivity was measured again after 24 hours, and the difference between the two measurements was calculated and recorded as the freeze-thawed sugarcane conductivity.

[0031] The experimental results are shown in Table 1 and Figure 1 As shown, compared with the conductivity of fresh sugarcane (45.0 μS / cm), the conductivity of sugarcane after freeze-thaw treatment increased to 82.5 μS / cm, but the conductivity of sugarcane after freeze-drying treatment increased significantly to 856.3 μS / cm, indicating that freeze-drying treatment caused the greatest damage to sugarcane cells. Figure 1 Scanning electron microscopy images showed that the surface of freeze-dried sugarcane had cavities and cracks, with a significantly greater degree of change than that of room temperature and freeze-thaw treatments. This verified the conclusion drawn from the conductivity comparison, namely that freeze-drying is more conducive to the extraction of cell contents than room temperature and freeze-thaw treatments.

[0032] Table 1. Electrical conductivity of sugarcane under different treatments

[0033]

[0034] Example 2: Effects of different extraction methods on the extraction rate and antioxidant activity of sugarcane polyphenols

[0035] 1. Extraction steps of sugarcane polyphenols in different groups

[0036] Group 1: Take 5g of freeze-dried sugarcane bagasse, add 150ml of deionized water, and pulse-treat under an electric field strength of 2KV / cm and a pulse count of 60. Then filter to obtain pulse-treated sugarcane bagasse 1. Add 150ml of 60% ethanol solution to pulse-treated sugarcane bagasse 1, and extract with ultrasonic assistance at 30℃ for 60min at an ultrasonic power of 120W. Centrifuge, filter, and concentrate under vacuum to obtain concentrate 1; freeze-dry concentrate 1 at -80℃ to obtain sugarcane polyphenol extract 1.

[0037] Group 2: Take 5g of freeze-dried sugarcane bagasse, add 150ml of ethanol solution, and extract with ultrasonic assistance at 60℃ for 90min (ultrasonic power 120W). Centrifuge and filter, then concentrate under vacuum to obtain concentrate 2; freeze-dry concentrate 2 at -80℃ to obtain sugarcane polyphenol extract 2.

[0038] Group 3: Take 5g of freeze-dried sugarcane bagasse, add 150ml of ethanol solution, and microwave extract for 90min at 300W. Centrifuge and filter, then concentrate under vacuum to obtain concentrate 3; 2. Freeze-dry concentrate 3 at -80℃ to obtain sugarcane polyphenol extract 3.

[0039] Group 4: Take 5g of freeze-dried sugarcane bagasse, add 150ml of deionized water, and pulse-treat under an electric field strength of 2KV / cm and a pulse number of 60. Then filter to obtain pulse-treated sugarcane bagasse 1. Add 150ml of 60% ethanol solution to the pulse-treated sugarcane bagasse obtained in step 1, and extract at 60℃ for 90min. Centrifuge and filter, and concentrate under vacuum to obtain concentrate 4. Freeze-dry concentrate 4 at -80℃ to obtain sugarcane polyphenol extract 4.

[0040] The surface morphology of sugarcane bagasse after treatment was compared under a scanning electron microscope.

[0041] 2. Determination of the physicochemical properties of the obtained sugarcane polyphenol extract

[0042] (1) Observation of the appearance and morphology of sugarcane samples

[0043] Sugarcane samples were adhered to the sample stage and placed in a vacuum spraying instrument to deposit a layer of conductive platinum film. The morphology of the polysaccharides was then observed using a scanning electron microscope JSM-7500F (JEOL, Japan) under a high voltage of 5kV.

[0044] (2) Determination of total phenol content

[0045] Folin-Ciocalteu method: Preparation of gallic acid standard curve. Accurately weigh gallic acid standard and dissolve it in deionized water to prepare a 0.1 mg / ml stock solution, which should be stored in the dark for later use. Dilute the 0.1 mg / ml stock solution to prepare working solutions of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / ml, and store them in the dark for later use. Add 0.5 ml of the working solution to a test tube, add 0.5 ml of Folin-Ciocalteu reagent, react in the dark for 5 min, then add 0.75 ml of 20% Na₂CO₃ and 3.75 ml of deionized water, mix well, and react at 37°C for 20 min. After the reaction, measure the absorbance at 760 nm. Repeat three parallel experiments. Plot the gallic acid standard curve with gallic acid concentration on the x-axis and absorbance on the y-axis.

[0046] The regression equation obtained is: Y = 0.0061X + 0.0597

[0047] The extracted sugarcane polyphenol extract was prepared into a solution of appropriate concentration and stored in the dark for later use. 0.5 ml of the polyphenol extract solution was taken and the absorbance of the sample was measured at 760 nm using the Folin-Ciocalteu method. Three parallel experiments were repeated. The polyphenol content in the sample was calculated based on the regression equation described above. The polyphenol content is calculated using the following formula:

[0048] Polyphenol content % = (Y - 0.0597) * 100% / (0.0061 * C * 1000)

[0049] Y - Absorbance value, C - Sample concentration (mg / ml)

[0050] (3) Antioxidant activity - DPPH free radical scavenging capacity determination

[0051] Accurately weigh 0.0040 g of DPPH and dissolve it in 5 ml of anhydrous ethanol to prepare a 2 mM DPPH stock solution. Store at 0-4°C protected from light. Dilute the DPPH stock solution 10 times with anhydrous ethanol to prepare a working solution, which should be used within 3.5 hours.

[0052] The extracted sugarcane polyphenols were prepared into solutions of different concentrations and stored in the dark for later use. 100 μl of each concentration of sugarcane polyphenol sample was added to a 96-well plate, followed by 100 μl of DPPH-anhydrous ethanol solution. The mixture was thoroughly incubated at room temperature (25°C) for 30 min in the dark. Using deionized water as a blank control, the absorbance at 517 nm was measured, and the DPPH scavenging activity and IC50 value were calculated.

[0053]

[0054] A1 represents the absorbance of the reaction system of sugarcane polyphenol solution and DPPH-ethanol solution.

[0055] (100 μL sugarcane molasses polyphenol sample + 100 μL DPPH-anhydrous ethanol solution)

[0056] A2 represents the absorbance of sugarcane polyphenol solution and anhydrous ethanol.

[0057] (100 μL sugarcane polyphenol solution + 100 μL anhydrous ethanol solution)

[0058] A3 represents the absorbance of deionized water and DPPH-anhydrous ethanol solution.

[0059] (100 μL deionized water + 100 μL DPPH-anhydrous ethanol solution)

[0060] A4 represents the absorbance of a deionized water-anhydrous ethanol solution.

[0061] (100 μL deionized water + 100 μL anhydrous ethanol solution)

[0062] 3. Experimental Results

[0063] like Figure 2 The scanning electron microscopy images show that the sugarcane cell surface was most severely damaged in the pulsed ultrasound group. As shown in Table 2, the total phenol content obtained after freeze-drying and different extraction methods showed significant differences. The total phenol content extracted by pulsed ultrasound was the highest, reaching 156.23 mg / 100g. At the same time, the sugarcane polyphenols obtained by this method had the strongest antioxidant energy and the lowest DPPH free radical scavenging IC50 value, which was 0.86 mg / mL.

[0064] Table 2. Total phenolic content and DPPH free radical scavenging ability of polyphenol extracts obtained in the examples and comparative examples.

[0065]

[0066] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A method for extracting sugarcane polyphenols, characterized in that, Including the following steps: S1: Fresh sugarcane is crushed into residue and then freeze-dried at low temperature to obtain freeze-dried sugarcane residue; S2: Mix freeze-dried sugarcane bagasse with ethanol and pre-treat with a pulsed electric field; S3: The freeze-dried sugarcane bagasse pretreated with pulsed electric field is ultrasonically extracted to obtain an extract; S4: Centrifuge the extract and filter it. Collect the filtrate and concentrate it. Freeze-dry the concentrate to obtain sugarcane polyphenol extract. The pulse processing conditions in step S2 are: pulse intensity of 1~5KV / cm, pulse width of 8~12 μs, and pulse number of 10~100. The conditions for ultrasonic treatment in step S3 are: temperature 30~60℃, power 40~200W, and duration 30~90min.

2. The method for extracting sugarcane polyphenols according to claim 1, characterized in that, In step S1, the low-temperature freeze-drying temperature is -100~-70℃.

3. The method for extracting sugarcane polyphenols according to claim 1, characterized in that, The volume of the crushed bagasse in step S1 shall not exceed 1 mm. 3 .

4. The method for extracting sugarcane polyphenols according to claim 1, characterized in that, In step S2, the volume ratio of sugarcane bagasse to ethanol is 1:(10~50).

5. The method for extracting sugarcane polyphenols according to claim 1, characterized in that, The centrifugation conditions in step S4 are: 2000~5000g, 5~10min.

6. The method for extracting sugarcane polyphenols according to claim 1, characterized in that, The concentration temperature in step S4 is 40~45℃.

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