Method for fractionally extracting citrus peel polysaccharide and flavone by synergistic effect of pulsed electric field and cellulase

Abstract

This invention discloses a method for fractional extraction of polysaccharides and flavonoids from dried tangerine peel using a pulsed electric field synergistic with cellulase. The method includes: first, pulverizing and sieving the dried tangerine peel; then, completely immersing the tangerine peel powder in distilled water; treating with a pulsed electric field; extracting in a water bath; filtering to obtain polysaccharides; and then subjecting the filter residue to a secondary extraction using cellulase to obtain highly active flavonoid components (hesperidin), while simultaneously collecting and recovering ethanol. This method is simple, highly efficient, and allows for solvent reuse. The pulsed electric field disrupts the cell walls of the dried tangerine peel, promoting polysaccharide dissolution and increasing the accessibility of the raw material for enzymatic hydrolysis, thus enhancing the subsequent extraction of hesperidin. This achieves highly efficient extraction of polysaccharides and flavonoid components (hesperidin) from dried tangerine peel. The resulting extract exhibits strong antioxidant, hypoglycemic, and lipid-lowering activities, showing potential application value in the fields of health products and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction, specifically involving a method for graded extraction of tangerine peel polysaccharides and flavonoids by cellulase synergistic pulsed electric field extraction. Background Technology

[0002] Dried tangerine peel (Chenpi) is the dried, mature peel of the citrus fruit (Citrus reticulata) and its cultivated varieties, belonging to the Rutaceae family. It is a typical food and medicine homology substance, possessing properties such as regulating qi and strengthening the spleen, and resolving dampness and phlegm. The main chemical components of dried tangerine peel include flavonoids, polysaccharides, volatile oils, and alkaloids, among which flavonoids are the most important active ingredients and a key focus of research. The flavonoid components in dried tangerine peel are mainly polymethoxyflavonoids (such as hesperidin and naringin) and flavonoid glycosides (such as hesperidin and naringin), with hesperidin having the highest content and possessing various biological functions, including antibacterial, anticancer, anti-inflammatory, antioxidant, and antidiabetic effects. Dried tangerine peel polysaccharides are considered a new generation of prebiotics, promoting the growth of probiotics, regulating intestinal flora structure, and playing an important role in improving gastrointestinal and cardiovascular diseases. Therefore, how to efficiently and cost-effectively utilize dried tangerine peel resources for its industrial application is of great significance.

[0003] Traditional methods for extracting flavonoids and polysaccharides from dried tangerine peel still rely on water extraction and organic solvent extraction. However, flavonoids are mainly distributed in the thin-walled cells of the mesocarp, and dried tangerine peel contains a large amount of pectin, which is tightly cross-linked with many active ingredients, increasing the difficulty of extraction, resulting in poor extraction efficiency and resource waste. Furthermore, these single extraction methods can only extract one functional active ingredient, and the organic solvents used, such as acetone and ethyl acetate, are highly volatile, causing serious industrial pollution. With the development of science and technology, some emerging extraction technologies have begun to emerge. Pulsed electric fields (PEF) are a novel non-thermal processing technology that has less impact on food components compared to traditional thermal processing, effectively reducing the loss of nutrients during processing. Its principle mainly utilizes the electroporation effect; by applying a pulsed electric field of a certain intensity to the object being processed, the integrity of the cell wall structure is disrupted, significantly increasing cell membrane permeability and improving extraction yield. For example, invention patent CN116422003A discloses a method for extracting theophylline from Jianghua bitter tea using a high-voltage pulsed electric field; the process is simple, and the extracted product has a high extraction rate. Tan et al. (Tan MJ, Li Y, Zhao S, Yue F, Cai D, Wu J, Han Z. 2024. Synergistic ultrasound pulsed electric field extraction of litchi peelpolyphenols and determination of their properties[J].International Journal of Biological Macromolecules (2024, 260: 129613.) The effect of pulsed electric field combined with ultrasound on the recovery rate of polyphenols from litchi peel was studied. The total phenol content of the obtained sample was 2.30 times that of the sample extracted by traditional hot water extraction. On the other hand, enzymatic hydrolysis is used in plant extraction due to its mild conditions and high selectivity. Chinese invention patent application CN107435059A discloses a method for extracting hesperidin by enzymatic hydrolysis using cellulase and pectinase. This method degrades cell wall components through enzymatic hydrolysis, increasing the hesperidin yield to 4.28%~4.4%. However, the enzymatic hydrolysis reaction of this technology needs to be carried out at 50~60℃ for 2~3 hours, followed by a 2~3 hour alcohol extraction process, with an overall process cycle of 4~6 hours and low production efficiency. In addition, the amount of compound enzyme added is as high as 2.5%~4.5%, which not only significantly increases the production cost, but also only targets the single component of hesperidin for extraction, failing to achieve the graded recovery of multiple components in tangerine peel. In summary, while pulsed electric field technology can efficiently break down cell walls, it is difficult to specifically degrade the cross-linked network formed by pectin and cellulose in tangerine peel when used alone. Enzymatic hydrolysis technology can directionally degrade cell walls, but it suffers from problems such as long reaction time, high cost, and limited extraction of single components.

[0004] Currently, there is no technology that combines the non-thermal crushing advantages of pulsed electric fields with the directional degradation characteristics of cellulase to construct an efficient process suitable for the graded extraction of polysaccharides and flavonoids from tangerine peel. Therefore, developing a green, efficient extraction technology for active ingredients from tangerine peel that can achieve synergistic recovery of multiple components has become an urgent technical need. Summary of the Invention

[0005] In order to overcome the shortcomings and defects of the existing technology, the purpose of this invention is to provide a method for preparing highly active tangerine peel polysaccharides and flavonoids with high extraction efficiency, mild extraction conditions, simple steps, and green environmental protection. The method uses pulsed electric field to synergistically prepare tangerine peel extract with cellulase, reducing the loss of active substances, and has potential application value in the fields of health products and functional foods.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides a method for fractional extraction of polysaccharides and flavonoids from tangerine peel using a pulsed electric field-assisted cellulase extraction process, comprising the following steps: 1) Raw material preparation: Grind and sieve the dried tangerine peel to obtain dried tangerine peel powder, and set aside; 2) Pulsed electric field (PEF) treatment: The tangerine peel powder is mixed evenly with distilled water and subjected to pulsed electric field treatment. Then, it is extracted and filtered in a water bath to obtain filtrate and residue. The filtrate is concentrated, precipitated with alcohol, washed, and freeze-dried to obtain polysaccharide powder. The residue is reserved for later use. 3) Enzymatic hydrolysis: The filter residue is mixed with ethanol solution and soaked. Cellulase is added to carry out the enzymatic hydrolysis reaction. The enzyme is inactivated to obtain the enzymatic hydrolysate. The enzymatic hydrolysate is filtered and concentrated. Ethanol is collected and recovered. The concentrated solution is freeze-dried to obtain hesperidin powder, which is the flavonoid component. 4) Ethanol recovery and reuse: The concentration of recovered ethanol is determined by a densitometer. Based on the measurement results, an appropriate amount of anhydrous ethanol or water is added to adjust it to the required concentration for the next batch of extraction.

[0007] Preferably, in step 1), the dried tangerine peel powder is passed through a 60-80 mesh sieve.

[0008] Preferably, in step 2), the ratio of dried tangerine peel powder to distilled water is 1:10~1:20 g / mL.

[0009] Preferably, in step 2), the pulsed electric field processing conditions are: electric field strength 3.5~5.5kV / cm, pulse number 800~1200 times, pulse width 10~20μs, and frequency 20~40Hz.

[0010] Preferably, in step 2), the water bath temperature is 45~55℃ and the water bath time is 40~60 min.

[0011] Preferably, in step 2), the concentration is carried out at 40~50℃ to 10%~20% of the original volume; the alcohol precipitation is carried out using anhydrous ethanol; the rinsing solvent is distilled water; the freeze-drying temperature is -60~-40℃ and the freeze-drying time is 20~24h.

[0012] Preferably, in step 3), the concentration of ethanol, by volume percentage, is 35% to 45%, the ratio of filter residue to ethanol solution is 1:8 to 1:10 g / mL, and the soaking time is 15 to 25 min.

[0013] Preferably, in step 3), the cellulase activity is 18000~22000U / mL, the amount of cellulase used accounts for 0.8%~1.6% of the mass of tangerine peel powder, the enzymatic hydrolysis time is 40~60min, and the enzymatic hydrolysis temperature is 45~55℃.

[0014] Preferably, in step 3), the enzyme inactivation temperature is 90~99℃ and the enzyme inactivation time is 3~5min.

[0015] Preferably, in step 3), the concentration is carried out at 40~50℃ to 20%~25% of the original volume, the freeze-drying temperature is -60~-40℃, and the freeze-drying time is 20~24h.

[0016] This invention also provides the application of the above-mentioned method for graded extraction of tangerine peel polysaccharides and flavonoids by cellulase in the preparation of functional foods and health products related to tangerine peel polysaccharides or flavonoids.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0018] (1) This invention combines PEF pretreatment with cellulosic hydrolysis technology. PEF pretreatment can efficiently destroy the cell wall structure of tangerine peel, which not only promotes the dissolution of water-soluble polysaccharides, but more importantly, greatly increases the accessibility of cellulase to cell wall substrates. The two produce a synergistic effect, thereby significantly shortening the total extraction time while simultaneously increasing the yield of polysaccharides and hesperidin.

[0019] (2) This invention addresses the differences in the physicochemical properties of polysaccharides (water-soluble) and hesperidin (water-insoluble) in tangerine peel, as well as the differences in their distribution within the tissue. It employs a sequential extraction method: first, polysaccharides are extracted using mild PEF conditions combined with hot water, and the degree of cell wall damage is controlled by the electric field strength to minimize premature and excessive dissolution of hesperidin; then, cellulase is specifically used to further deconstruct the cell wall network, efficiently releasing hesperidin. This process enables the sequential and efficient acquisition of two high-value products from the same raw material, greatly enhancing the comprehensive utilization value and industrial added value of tangerine peel resources.

[0020] (3) The polysaccharide extract extracted by the present invention has good antioxidant properties. The antioxidant activity, hypoglycemic activity and lipid-lowering activity of the hesperidin extract are significantly better than those of the single enzymatic hydrolysis product. Moreover, the extraction process uses water and ethanol as the main solvents, the solvent recovery is simple and energy consumption is low, no toxic and harmful reagents are introduced, the product has high safety, and it can be applied to functional foods and health products, etc., with broad market application prospects. Attached Figure Description

[0021] Figure 1 The images are scanning electron microscope (SEM) images of dried tangerine peel used in Embodiment 1 and Comparative Examples 1-3 of the present invention.

[0022] Figure 2 The graphs show the ABTS free radical scavenging rate of the polysaccharides prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.

[0023] Figure 3 The graphs show the DPPH free radical scavenging rate of hesperidin prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.

[0024] Figure 4 The hesperidin prepared in Examples 1-3 and Comparative Examples 1-5 of this invention α -Graph showing the effect of glucosidase inhibition.

[0025] Figure 5 The flowchart of the method for graded extraction of tangerine peel polysaccharides and flavonoids by cellulase in synergistic pulsed electric field provided by the present invention is shown. Detailed implementation mode

[0026] To better understand the present invention, the following further describes the present invention in conjunction with the accompanying drawings and embodiments, but the implementation mode of the present invention is not limited thereto.

[0027] Figure 5 The flowchart of the method for fractionated extraction of tangerine peel polysaccharide and flavonoid by pulsed electric field in cooperation with cellulase provided by the present invention is shown.

[0028] In the embodiments of the present invention, the tangerine peel is Guang tangerine peel. In the embodiments of the present invention, the pulsed electric field equipment selects the SY-500 type pulsed electric field equipment of Guangzhou Paihu Technology Co., Ltd. Figure 1 The scanning electron microscope images of the tangerine peel used in Example 1 and Comparative Examples 1-3 of the present invention are shown. 500X is the magnification of 500 times, and 1000X is the magnification of 1000 times.

[0029] Example 1 A method for fractionated extraction of tangerine peel polysaccharide and flavonoid by pulsed electric field in cooperation with cellulase includes the following steps: Raw material preparation: The tangerine peel is crushed and sieved through a 70-mesh sieve.

[0030] Pulsed electric field treatment: Mix 10 g of tangerine peel powder with distilled water according to a solid-liquid ratio of 1:15 g / mL, put it into the pulsed electric field equipment for treatment, set the pulsed electric field intensity to 4.5 kV / cm, the number of pulses to 1200 times, the pulse width to 15 μs, and the electric field frequency to 30 Hz. After treatment, extract in a water bath at 50 °C for 50 min, filter to obtain the filtrate and the residue, and reserve the residue; Rotate and evaporate the filtrate at 45 °C to concentrate it to 10% of the original volume. Subsequently, add 2 times the volume of absolute ethanol to the concentrated solution for alcohol precipitation, stand at 4 °C for 2 h, take the precipitate, wash it twice with water, and freeze-dry it at -50 °C for 22 h to obtain the polysaccharide powder, with a yield of 25.95%. The DPPH and ABTS radical scavenging rates are 88.22% and 90.08% respectively.

[0031] Enzymatic hydrolysis treatment: Mix the residue with a 40% (v / v) ethanol solution according to a solid-liquid ratio of 1:9 g / mL, soak it for 20 min, then add 1.2% of cellulase (enzyme activity 20000 U / mL) based on the mass of the tangerine peel powder, carry out enzymatic hydrolysis reaction at 50 °C for 50 min, and inactivate the enzyme at 96 °C for 4 min to obtain the enzymatic hydrolysis solution, and filter to obtain the filtrate. Rotate and evaporate the filtrate at 45 °C to concentrate it to 20% of the original volume, synchronously collect and recycle the ethanol, and reuse it for the extraction of hesperidin (ethanol recovery rate 86.11%). Transfer the concentrated solution to a freeze dryer, and freeze-dry it at -50 °C for 22 h to obtain the hesperidin powder, with a yield of 9.74%. The DPPH and ABTS radical scavenging rates are 89.62% and 87.50% respectively, α The inhibition rate of α-glucosidase is 82.93%.

[0032] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 44.28% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0033] Example 2 A method for fractional extraction of polysaccharides and flavonoids from tangerine peel using a pulsed electric field-assisted cellulase extraction process includes the following steps: Raw material preparation: Powder the dried tangerine peel and pass it through a 60-mesh sieve.

[0034] Pulsed electric field treatment: 10g of dried tangerine peel powder was mixed with distilled water at a ratio of 1:10g / mL and placed in a pulsed electric field device for treatment. The pulsed electric field strength was set to 3.5kV / cm, the number of pulses was 800, the pulse width was 10μs, and the electric field frequency was 20Hz. After treatment, the mixture was extracted in a 45℃ water bath for 40min. The filtrate and residue were obtained by filtration, and the residue was reserved. The filtrate was concentrated by rotary evaporation at 40℃ to 15% of its original volume. Then, two volumes of anhydrous ethanol were added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2h, and the precipitate was washed twice with water and freeze-dried at -60℃ for 20h to obtain polysaccharide powder with a yield of 23.07%. The DPPH and ABTS free radical scavenging rates were 81.47% and 82.36%, respectively.

[0035] Enzymatic hydrolysis: The filter residue was mixed with 35% (v / v) ethanol solution at a material-to-liquid ratio of 1:8 g / mL and soaked for 15 min. Then, 0.8% (by weight of tangerine peel powder) of cellulase (enzyme activity 18000 U / mL) was added, and the enzymatic hydrolysis reaction was carried out at 45℃ for 40 min. The enzyme was then inactivated at 90℃ for 3 min to obtain the enzymatic hydrolysate, which was filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 40℃ to 23% of its original volume, and the ethanol was collected and recovered simultaneously. This was reused for hesperidin extraction (ethanol recovery rate 81.25%). The concentrate was transferred to a freeze dryer and freeze-dried at -60℃ for 20 h to obtain hesperidin powder with a yield of 8.21%. The DPPH and ABTS free radical scavenging rates were 80.09% and 78.39%, respectively. α - The glucosidase inhibition rate was 74.14%.

[0036] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 41.93% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0037] Example 3 A method for fractional extraction of polysaccharides and flavonoids from tangerine peel using a pulsed electric field-assisted cellulase extraction process includes the following steps: Raw material preparation: Powder the dried tangerine peel and pass it through an 80-mesh sieve.

[0038] Pulsed electric field treatment: 10g of dried tangerine peel powder was mixed with distilled water at a ratio of 1:20g / mL and placed in a pulsed electric field device for treatment. The pulsed electric field strength was set to 5.5kV / cm, the number of pulses was 1000, the pulse width was 20μs, and the electric field frequency was 40Hz. After treatment, the mixture was extracted in a water bath at 55℃ for 60min. The filtrate and residue were obtained by filtration, and the residue was reserved. The filtrate was concentrated by rotary evaporation at 50℃ to 20% of its original volume. Then, two volumes of anhydrous ethanol were added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2h, and the precipitate was washed twice with water and freeze-dried at -40℃ for 24h to obtain polysaccharide powder with a yield of 26.81%. The DPPH and ABTS free radical scavenging rates were 90.13% and 92.71%, respectively.

[0039] Enzymatic hydrolysis: The filter residue was mixed with 45% (v / v) ethanol solution at a material-to-liquid ratio of 1:10 g / mL and soaked for 25 min. Then, 1.6% (by weight of dried tangerine peel powder) of cellulase (enzyme activity 22000 U / mL) was added, and the enzymatic hydrolysis reaction was carried out at 55℃ for 60 min. The enzyme was then inactivated at 99℃ for 5 min to obtain the enzymatic hydrolysate, which was filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 50℃ to 25% of its original volume, and the ethanol was collected and recovered simultaneously. This was reused for hesperidin extraction (ethanol recovery rate 87.50%). The concentrate was transferred to a freeze dryer and freeze-dried at -40℃ for 24 h to obtain hesperidin powder with a yield of 8.01%. The DPPH and ABTS free radical scavenging rates were 78.64% and 76.43%, respectively. α - The glucosidase inhibition rate was 72.87%.

[0040] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 46.66% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0041] Comparative Example 1 Raw material preparation: Powder the dried tangerine peel and pass it through a 70-mesh sieve.

[0042] Polysaccharide extraction: 10g of dried tangerine peel powder was mixed with distilled water at a ratio of 1:15g / mL and extracted in a 50℃ water bath for 50min. The mixture was filtered to obtain a filtrate and a residue, which was then set aside. The filtrate was concentrated by rotary evaporation at 45℃ to 10% of its original volume. Two volumes of anhydrous ethanol were then added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2h, and the precipitate was washed twice with water and freeze-dried at -50℃ for 22h to obtain polysaccharide powder, with a yield of 16.77%. The DPPH and ABTS free radical scavenging rates were 64.91% and 67.42%, respectively.

[0043] Hesperidin extraction: The filter residue was mixed with 40% (v / v) ethanol solution at a material-to-liquid ratio of 1:9 g / mL and soaked for 20 min. The mixture was then incubated in a water bath at 50℃ for 50 min, followed by filtration to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 45℃ to 20% of its original volume, with ethanol collected and recovered simultaneously. This concentrated filtrate was reused for hesperidin extraction (ethanol recovery rate 83.33%). The concentrate was transferred to a freeze dryer and freeze-dried at -50℃ for 22 h to obtain hesperidin powder, with a yield of 5.03%. The DPPH and ABTS free radical scavenging rates were 59.11% and 58.36%, respectively. α - The glucosidase inhibition rate was 52.73%.

[0044] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 42.85% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0045] Comparative Example 2 Raw material preparation: Powder the dried tangerine peel and pass it through a 70-mesh sieve.

[0046] Polysaccharide extraction: 10g of dried tangerine peel powder was mixed with distilled water at a material-to-liquid ratio of 1:15g / mL and treated in a pulsed electric field device. The pulsed electric field strength was set to 4.5kV / cm, the number of pulses was 1200, the pulse width was 15μs, and the electric field frequency was 30Hz. After treatment, the mixture was extracted in a 50℃ water bath for 50min. The filtrate and residue were obtained by filtration, and the residue was reserved. The filtrate was concentrated by rotary evaporation at 45℃ to 10% of its original volume. Then, two volumes of anhydrous ethanol were added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2h, and the precipitate was washed twice with water and freeze-dried at -50℃ for 22h to obtain polysaccharide powder with a yield of 25.95%. The DPPH and ABTS free radical scavenging rates were 88.22% and 90.08%, respectively.

[0047] Hesperidin extraction: The filter residue was mixed with 40% (v / v) ethanol solution at a material-to-liquid ratio of 1:9 g / mL and soaked for 20 min. The mixture was then incubated in a water bath at 50℃ for 50 min, followed by filtration to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 45℃ to 20% of its original volume, with ethanol collected and recovered simultaneously. This concentrated filtrate was reused for hesperidin extraction (ethanol recovery rate 81.94%). The concentrate was transferred to a freeze dryer and freeze-dried at -50℃ for 22 h to obtain hesperidin powder, with a yield of 4.76%. The DPPH and ABTS free radical scavenging rates were 57.74% and 55.41%, respectively. α - The glucosidase inhibition rate was 51.08%.

[0048] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 41.54% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0049] Comparative Example 3 Raw material preparation: Powder the dried tangerine peel and pass it through a 70-mesh sieve.

[0050] Polysaccharide extraction: 10g of dried tangerine peel powder was mixed with distilled water at a ratio of 1:15g / mL and extracted in a 50℃ water bath for 50min. The mixture was filtered to obtain a filtrate and a residue, which was then set aside. The filtrate was concentrated by rotary evaporation at 45℃ to 10% of its original volume. Two volumes of anhydrous ethanol were then added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2h, and the precipitate was washed twice with water and freeze-dried at -40℃ for 24h to obtain polysaccharide powder, with a yield of 16.77%. The DPPH and ABTS free radical scavenging rates were 64.91% and 67.42%, respectively.

[0051] Hesperidin extraction: The filter residue was mixed with 40% (v / v) ethanol solution at a material-to-liquid ratio of 1:9 g / mL and soaked for 20 min. Then, 1.2% (by weight of dried tangerine peel powder) of cellulase (enzyme activity 20000 U / mL) was added, and the enzymatic hydrolysis was carried out at 50℃ for 50 min. The enzyme was then inactivated at 96℃ for 4 min to obtain the enzymatic hydrolysate, which was filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 45℃ to 20% of its original volume, and the ethanol was collected and recovered simultaneously. This was reused for hesperidin extraction (ethanol recovery rate 77.78%). The concentrate was transferred to a freeze dryer and freeze-dried at -50℃ for 22 h to obtain hesperidin powder with a yield of 6.74%. The DPPH and ABTS free radical scavenging rates were 68.82% and 67.12%, respectively. α - The glucosidase inhibition rate was 63.57%.

[0052] Ethanol recovery and reuse: The concentration of recovered ethanol is determined by a densitometer. The ethanol concentration is 40.00% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water is added to adjust it to the required concentration for the next batch of extraction.

[0053] Comparative Example 4 Raw material preparation: Powder the dried tangerine peel and pass it through a 70-mesh sieve.

[0054] Tangerine peel powder was mixed with 40% (v / v) ethanol solution at a material-to-liquid ratio of 1:9 g / mL and soaked for 20 min. Then, 1.2% (by weight of tangerine peel powder) of cellulase (enzyme activity 20000 U / mL) was added, and the mixture was enzymatically hydrolyzed at 50℃ for 50 min, followed by enzyme inactivation at 96℃ for 4 min to obtain the enzymatic hydrolysate. The hydrolysate was then filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 45℃ to 20% of its original volume. Ethanol was collected and recovered and reused for hesperidin extraction (ethanol recovery rate 79.17%). The concentrate was transferred to a freeze dryer and freeze-dried at -50℃ for 22 h to obtain a mixed powder. The polysaccharide content and hesperidin content were determined to be 12.15% and 7.13%, respectively. The DPPH and ABTS free radical scavenging rates of the mixture were 73.39% and 71.24%, respectively. α - The glucosidase inhibition rate was 67.19%.

[0055] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 40.71% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0056] Comparative Example 5 Raw material preparation: Powder the dried tangerine peel and pass it through a 70-mesh sieve.

[0057] Enzymatic hydrolysis: 10g of dried tangerine peel powder was mixed with 40% (v / v) ethanol solution at a material-to-liquid ratio of 1:9g / mL and soaked for 20min. Then, 1.2% (by weight of the tangerine peel powder) of cellulase (enzyme activity 20000U / mL) was added, and the enzymatic hydrolysis reaction was carried out at 50℃ for 50min. The enzyme was then inactivated at 96℃ for 4min to obtain the hydrolysate. The filtrate was obtained by filtration, and the residue was reserved. The filtrate was concentrated by rotary evaporation at 45℃ to 20% of its original volume. Ethanol was collected and recovered simultaneously and reused for hesperidin extraction (ethanol recovery rate 79.17%). The concentrate was transferred to a freeze dryer and freeze-dried at -50℃ for 22h to obtain hesperidin powder with a yield of 7.13%. The DPPH and ABTS free radical scavenging rates were 72.06% and 70.54%, respectively. α - The inhibition rate of glucosidase was 66.68%.

[0058] Pulsed electric field treatment: The filter residue was mixed with distilled water at a material-to-liquid ratio of 1:15 g / mL and placed in a pulsed electric field device for treatment. The pulsed electric field strength was set to 4.5 kV / cm, the number of pulses was 1200, the pulse width was 15 μs, and the electric field frequency was 30 Hz. After treatment, the residue was extracted in a 50℃ water bath for 50 min and filtered to obtain the filtrate. The filtrate was concentrated by rotary evaporation at 45℃ to 10% of its original volume. Then, two volumes of anhydrous ethanol were added to the concentrate for alcohol precipitation. The mixture was allowed to stand at 4℃ for 2 h, and the precipitate was washed twice with water and freeze-dried at -50℃ for 22 h to obtain polysaccharide powder with a yield of 10.26%. The DPPH and ABTS free radical scavenging rates were 42.99% and 47.29%, respectively.

[0059] Ethanol recovery and reuse: The concentration of recovered ethanol was determined by a densitometer. The ethanol concentration was 40.71% (v / v). Based on the measurement results, an appropriate amount of anhydrous ethanol or water was added to adjust it to the required concentration for the next batch of extraction.

[0060] The tangerine peel extracts prepared in Examples 1-3 and Comparative Examples 1-5 were measured using the following method.

[0061] 1. Content Determination: The polysaccharide content was determined by the phenol-sulfuric acid method, and the hesperidin content was determined by HPLC. The HPLC instrument settings were as follows: the detection column was an Eclipse Plus C18 (100 mm × 4.6 mm, 3.5 µm), and the mobile phases were 0.2% acetic acid aqueous solution (pump A) and acetonitrile (pump B). Gradient elution was used, and the separation conditions were as follows: 0.00–3.00 min, pump B: 5%–18% (v / v); 3.00–20.00 min, pump B: 18%–22% (v / v); 20.00–25.00 min, pump B: 22%–50% (v / v); 25.00–30.00 min, pump B: 50%–70% (v / v); 30.00–31.00 min, pump B: 70%–5% (v / v); 31.00–38.00 min, pump B: 5%–5% (v / v). The flow rate was 1 mL / min, the detection wavelength was 283 nm, and the detection temperature was 25 °C.

[0062] In Examples 1-3 and Comparative Examples 1-5, after recovering ethanol, the ethanol was recycled and extracted twice more. The polysaccharide yield, hesperidin yield, ethanol recovery rate, polysaccharide yield per liter of water, and hesperidin content per milliliter of ethanol were statistically analyzed for each extraction. The results are shown in Table 1.

[0063] As shown in Table 1, compared with traditional water bath extraction (Comparative Example 1, blank control), after introducing pulsed electric field treatment and enzymatic hydrolysis in Examples 1-3, the polysaccharide yield of the first extraction increased from 16.77% to 23%-26%, and the hesperidin yield increased from 5.03% to 8%-9%. This indicates that electric field and enzymatic hydrolysis treatment can significantly improve the release efficiency of the target product. In addition, after two cycles of ethanol recycling in Example 1, the recovery rate was still over 80%, and the hesperidin yield remained above 90% of that of fresh ethanol, more than doubling the yield of Comparative Example 1 and saving 54% of ethanol costs; for the same polysaccharide yield, the water consumption of Comparative Example 1 was about 1.6 times that of Example 1, saving 37.5% of water. It is evident that the synergistic treatment of pulsed electric field and enzymatic hydrolysis significantly reduced the overall production cost, and the economic benefits after industrial scale-up are outstanding.

[0064] As shown in Table 1, Example 1 (with PEF and enzymatic hydrolysis), Comparative Example 2 (without enzymatic hydrolysis), and Comparative Example 3 (without PEF) showed a significant increase in polysaccharide and hesperidin yields. This result demonstrates that cellulase can specifically act on the cellulose network retained in the filter residue after polysaccharide extraction without interfering with the dissolved and separated polysaccharides. At the same time, PEF pretreatment not only directly promotes polysaccharide dissolution through electroporation but also breaks down cell wall structures, increasing the contact area between cellulase and substrate, thereby indirectly improving the hesperidin extraction efficiency.

[0065] Table 1. Effects of ethanol extraction and recovery on the extraction efficiency of tangerine peel polysaccharides and hesperidin.

[0066] As can be seen from Examples 1-3 and Comparative Example 4 in Table 1, the yields of polysaccharides and hesperidin in the mixture extracted by direct enzymatic hydrolysis were significantly lower than those in the fractional extraction. This may be related to the mutual interference between the polysaccharide and hesperidin systems in the mixed extraction, and the solvent system could not simultaneously accommodate the dissolution of both.

[0067] As shown in Table 1, compared with Examples 1-3, the yields of polysaccharides and hesperidin in Comparative Example 5 decreased significantly after reversing the order of "extracting polysaccharides first and then hesperidin". This is mainly attributed to the following: First, the initial alcohol extraction step may lead to the initial dissolution of some polysaccharides, and ethanol may change the properties of the material, hindering the full action of PEF on the cell wall in the subsequent process; second, the enzymatic hydrolysis step of hesperidin lacks the synergistic effect of electroporation due to the lack of PEF pretreatment, resulting in a decrease in extraction efficiency; in addition, ethanol and cellulase may have a synergistic effect in the early steps, which may have an adverse effect on the polysaccharide structure.

[0068] 2. Activity assay: DPPH scavenging effect: Prepare a 0.05 mg / mL DPPH solution, labeled as DPPH working solution. Add 2 mL of 0.55 mg / mL sample solution to 2 mL of DPPH working solution and incubate at room temperature in the dark for 30 min. Measure the absorbance value (A value) at a wavelength of 517 nm. Repeat the experiment 3 times, count the A value and calculate the DPPH free radical scavenging rate using Formula 1.

[0069] ABTS scavenging effect: 7 mmol / L ABTS solution and 4.9 mmol / L K2S2O8 solution were mixed at a volume ratio of 1:1 and reacted in the dark for 12 h. The solution was then diluted with ethanol to a absorbance of 0.70 ± 0.02 at 734 nm, and labeled as the ABTS working solution. 3 mL of the ABTS working solution was mixed with 1 mL of 0.55 mg / mL sample solution and reacted at room temperature for 10 min. The absorbance (A value) was measured at 734 nm. The experiment was repeated three times, and the A values ​​were statistically analyzed. The ABTS free radical scavenging rate was calculated using Formula 1.

[0070]

[0071] Where A0 is the absorbance value of the blank control group, i.e., ethanol is used instead of the sample solution; A1 is the absorbance value of the sample measurement group; and A2 is the absorbance value of the blank sample group, i.e., ethanol is used instead of the working solution.

[0072] The DPPH and ABTS free radical scavenging effects of the polysaccharides and hesperidin prepared in Examples 1-3 and Comparative Examples 1-5 are as follows: Figure 2 and Figure 3 As shown, the tangerine peel extract prepared by combining an electric field (3.5~5.5kV / cm) and enzymatic hydrolysis (with cellulase accounting for 0.8%~1.6% of the tangerine peel powder mass) under certain conditions exhibits better DPPH and ABTS free radical scavenging effects and better antioxidant properties compared to the control group, which corresponds to the changes in hesperidin and polysaccharide content in the tangerine peel extract. The combined use of electric field and enzymatic hydrolysis has better effects than traditional solvent extraction and single-method extraction of tangerine peel extract, thus enhancing the antioxidant properties of the tangerine peel extract.

[0073] α - Glucosidase inhibition effect: 0.5 mL of 1.5 mg / mL sample solution and 0.5 mL of... α Mix 1 U / ml glucosidase solution with water bath at 37°C for 10 min, then add 0.5 mL of 2.5 mmol / L PNPG substrate solution. Incubate at 37°C for 30 min to activate the enzyme. Stop the reaction by adding 2 mL of 1 mol / L Na₂CO₃ solution. Measure the absorbance (A value) at 405 nm. Repeat the experiment three times, and calculate the A value using Formula 2. α - Glucosidase inhibition rate.

[0074]

[0075] Where A0 is the absorbance value of the negative control group, i.e., PBS is used instead of the sample solution; A1 is the absorbance value of the sample measurement group; and A2 is the absorbance value of the sample background group, i.e., PBS is used instead of the sample background group. α - Glucosidase solution; A3 is the absorbance of the blank control group, i.e., using PBS instead of the sample solution and... α - Glucosidase solution.

[0076] Figure 4 The hesperidin prepared in Examples 1-3 and Comparative Examples 1-5 of this invention α -Graph showing the inhibition effect of glucosidase, illustrating the hesperidin prepared in Examples 1-3. α - The glucosidase inhibition rate is higher, and the scavenging effect is better than that of control groups 1-5. It has better hypoglycemic activity, which corresponds to the change in hesperidin content in tangerine peel extract. This proves that the hypoglycemic activity of the extract is greatly improved by the combined extraction method.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fractional extraction of polysaccharides and flavonoids from tangerine peel using a pulsed electric field-assisted cellulase extraction process, characterized in that... Includes the following steps: 1) Raw material preparation: Grind and sieve the dried tangerine peel to obtain dried tangerine peel powder, and set aside; 2) Pulsed electric field (PEF) treatment: Add tangerine peel powder to distilled water and mix evenly. Then, perform pulsed electric field treatment, followed by water bath extraction and filtration to obtain filtrate and residue. Concentrate the filtrate, precipitate with alcohol, rinse, and freeze-dry to obtain polysaccharide powder. Set aside the residue for later use. 3) Enzymatic hydrolysis: The filter residue is mixed with ethanol solution and soaked. Cellulase is added to carry out the enzymatic hydrolysis reaction. The enzyme is inactivated to obtain the enzymatic hydrolysate. The enzymatic hydrolysate is filtered and concentrated. Ethanol is collected and recovered. The concentrated solution is freeze-dried to obtain hesperidin powder, which is the flavonoid component. 4) Ethanol recovery and reuse: The concentration of recovered ethanol is determined by a densitometer. Based on the measurement results, an appropriate amount of anhydrous ethanol or water is added to adjust it to the required concentration for the next batch of extraction.

2. The method according to claim 1, characterized in that, In step 1), the dried tangerine peel powder is passed through a 60-80 mesh sieve.

3. The method according to claim 1, characterized in that, In step 2), the ratio of dried tangerine peel powder to distilled water is 1:10~1:20 g / mL.

4. The method according to claim 1, characterized in that, In step 2), the pulsed electric field processing conditions are: electric field strength 3.5~5.5kV / cm, pulse number 800~1200 times, pulse width 10~20μs, and frequency 20~40Hz.

5. The method according to claim 1, characterized in that, In step 2), the water bath temperature is 45~55℃ and the water bath time is 40~60min.

6. The method according to claim 1, characterized in that, In step 2), the concentration is carried out at 40~50℃ and concentrated to 10%~20% of the original volume; the alcohol precipitation is carried out using anhydrous ethanol; the rinsing solvent is distilled water; the freeze-drying temperature is -60 ~ -40℃ and the freeze-drying time is 20~24h.

7. The method according to claim 1, characterized in that, In step 3), the concentration of ethanol, in volume percentage, is 35%~45%, the ratio of filter residue to ethanol solution is 1:8~1:10 g / mL, and the soaking time is 15~25 min.

8. The method according to claim 1, characterized in that, Step 3), the cellulase activity is 18000~22000U / mL, the amount of cellulase used accounts for 0.8%~1.6% of the mass of tangerine peel powder, the enzymatic hydrolysis time is 40~60min, and the enzymatic hydrolysis temperature is 45~55℃.

9. The method according to claim 1, characterized in that, In step 3), the enzyme inactivation temperature is 90~99℃ and the enzyme inactivation time is 3~5min; the concentration is carried out at 40~50℃ and concentrated to 20%~25% of the original volume; the freeze-drying temperature is -60~-40℃ and the freeze-drying time is 20~24h.

10. The application of the method according to any one of claims 1 to 9 in the preparation of functional foods and health products related to tangerine peel polysaccharides or flavonoids.

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