Method for improving flavor and reducing bitterness of fructus aurantii immaturi extract by high pressure radio frequency cold plasma-enzyme coupling method

Abstract

The present application belongs to the field of preparation of medicinal and edible extract, and particularly relates to a method for improving flavor and reducing bitterness of fructus aurantii immaturi extract by high-pressure radio frequency cold plasma-enzyme coupling. The method comprises the following steps: adding water to fructus aurantii immaturi powder to swell, then adjusting pH, adding hydrolytic enzyme I and complex hydrolytic enzyme to the obtained slurry, and then hydrolyzing at constant temperature, then filtering, and placing the filtrate in a radio frequency cold plasma treatment device; controlling vacuum pressure to be 0.3±0.1 bar, and introducing argon; after radio frequency discharge treatment of the radio frequency cold plasma treatment device, fructus aurantii immaturi extract is obtained. By the method, bitter substances naringin and neohesperidin in fructus aurantii immaturi can be degraded or hydrolyzed, and the content of characteristic aroma components in fructus aurantii immaturi extract can be improved.

Description

Technical Field

[0001] This invention belongs to the field of preparation of medicinal and edible extracts, specifically involving a method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using a high-voltage radio frequency cold plasma-enzyme coupling method. Background Technology

[0002] Huazhou tangerine peel, also known as "hairy tangerine peel," is the dried outer peel of the immature or nearly mature fruit of the Huazhou pomelo or pomelo plant, belonging to the Rutaceae family. It has a long history and is commonly used to treat coughs due to wind-cold, nausea, and abdominal distension; it also has the effects of regulating qi, resolving phlegm, strengthening the stomach, and aiding digestion. Huazhou tangerine peel is a substance that is both a food and a traditional Chinese medicine.

[0003] Citrus reticulata extract has antitussive and expectorant effects, with its flavonoids and volatile oils exhibiting a synergistic effect; simultaneously, the extract also possesses excellent free radical scavenging capabilities. The main volatile organic compounds in Citrus reticulata are limonene, ... c - Terpinene, β - Geraniol, Nerolidol α -pinene, β Primarily composed of pinene and other compounds. Due to their natural properties, herbal essential oils and extracts are widely favored in the market. However, a drawback of Citrus reticulata extract is its high content of bitter substances, such as naringin and neohesperidin, which gives it a bitter taste. In addition, conventional hot water extraction or extraction combined with ultrasound and organic solvents can cause volatile aroma components to undergo oxidation, condensation, and polymerization reactions due to the high extraction temperature, the presence of organic solvents, or the long extraction time, resulting in a significant difference between the aroma and the actual aroma.

[0004] Cold plasma pretreatment is a novel non-thermal material treatment technology that uses high-energy particles to bombard the surface of materials, causing a series of physical and chemical changes such as etching, cell disruption, and grafting of chemical groups. This technology is characterized by high efficiency, environmental friendliness, low cost, short processing time, and large batch processing capacity. Its application in the extraction of bioactive substances, such as polysaccharides and pectin, as well as the degradation of toxic and harmful substances, such as patulin, has been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high voltage radio frequency cold plasma-enzyme coupling. This method can reduce the bitterness of Citrus reticulata extract, improve its taste, and enhance its aroma.

[0006] To address the aforementioned technical problems, this invention provides a method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling, comprising the following steps: 1) Pulverize the dried (moisture content ≤6%) Citrus reticulata peel to obtain Citrus reticulata peel powder; 2) Set the material-to-liquid mass ratio to 1:13~17; Add water to 5g of Citrus reticulata powder, and let the powder absorb water and swell for 3±0.5h, then adjust the pH to 6.5±0.2. Alternatively, after adding water to 5g of Citrus reticulata powder, adjust the pH to 6.5±0.2, and then allow the Citrus reticulata powder to absorb water and swell for 3±0.5h. Note: pH can be adjusted using 1N hydrochloric acid or 1N sodium hydroxide; 3) Add 500~1000U of hydrolytic enzyme I and 1000~2000U of compound hydrolytic enzyme to the liquid obtained in step 2). Under magnetic stirring, hydrolyze at a constant temperature of 40±2°C for 2±0.1h, then filter and place the filtrate in a radio frequency cold plasma treatment device. 4) Control the pressure (absolute pressure) to 0.3±0.1 bar using a vacuum pump, and introduce argon gas; The discharge parameters for controlling the radio frequency cold plasma treatment device are: voltage 60±3kV, frequency 25±1 kHz, discharge power 450±20 W, and treatment time 10~30min (radio frequency discharge 10~30min). The processed Citrus reticulata extract was obtained.

[0007] As an improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention: after the processing time of step 4) is reached, the radio frequency discharge is turned off, the vacuum pump is turned off, and the radio frequency cold plasma treatment device is allowed to return to normal pressure; the treated Citrus reticulata extract is obtained.

[0008] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention: in step 4): adjust the argon gas inlet flow rate to 5~15mL / min.

[0009] Note: The total volume of the cavity of the radio frequency cold plasma treatment device is approximately 10L.

[0010] As a preferred option: argon gas inlet flow rate of 10~15mL / min, and processing time (radio frequency discharge time) of 20~30min.

[0011] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention: in step 4): the distance between the radio frequency electrode and the liquid surface of the sample is adjusted to 9 ± 0.5 cm (that is, the vertical distance between the dielectric barrier discharge radio frequency high voltage electrode and the liquid surface of the sample is adjusted to 9 ± 0.5 cm).

[0012] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention, in step 3): hydrolytic enzyme I is naringinase; the complex hydrolytic enzyme is composed of at least two of β-glucosidase, β-galactosidase and cellulase; the content of each enzyme in the complex hydrolytic enzyme is 1000~2000U.

[0013] Naringinase is used to hydrolyze naringin, a bitter substance in tangerine peel, thereby reducing its bitterness.

[0014] The role of complex hydrolytic enzymes is to hydrolyze flavor precursors in tangerine peel that are bound by glycosidic bonds or cellulose, thereby releasing flavor substances and improving product flavor.

[0015] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling of the present invention: the complex hydrolytic enzyme is composed of 1 kU β-glucosidase, 1 kU β-galactosidase and 1 kU cellulase.

[0016] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention, in step 3): the filtration is filtration through a 1.2μm filter membrane.

[0017] As a further improvement to the method of enhancing the flavor and reducing the bitterness of Citrus reticulata extract by high-voltage radio frequency cold plasma-enzyme coupling method of the present invention, in step 1): Citrus reticulata powder can pass through a 200-mesh sieve.

[0018] This invention relates to a method for coupling cold plasma generated by high-voltage radio frequency with glycosidase during water extraction.

[0019] The method of this invention degrades or hydrolyzes the bitter substances naringin and neohesperidin in Citrus reticulata peel, while simultaneously increasing the content of characteristic aroma components. Because the high-voltage radio frequency cold isotope treatment operates at lower temperatures and under vacuum conditions, it avoids the destruction of easily oxidized components in the flavor compounds, thus preventing flavor alteration. The composite hydrolytic enzyme used releases non-volatile flavor precursors that exist in a glycosidic bond state, thereby enhancing the characteristic aroma of the final Citrus reticulata peel extract. The naringinase used also degrades the bitter substance naringin, reducing bitterness. The coupling of high-voltage radio frequency cold isotope treatment and hydrolytic enzyme treatment in this invention effectively reduces the content of naringin and hesperidin in the Citrus reticulata peel extract, thus reducing bitterness, and increases the relative content of characteristic flavor compounds such as olefins, thereby enhancing aroma, thus comprehensively improving the flavor of the Citrus reticulata peel extract. As a result, the final Citrus reticulata peel extract obtained by traditional methods such as boiling water extraction has lower bitterness and a stronger characteristic aroma.

[0020] The beneficial effects of this invention are mainly reflected in: This invention employs high-voltage radio frequency-generated cold plasma treatment combined with glycosidase treatment of Citrus reticulata extract, significantly reducing the bitter substances naringin and neohesperidin, effectively decreasing its bitterness. Simultaneously, it increases the characteristic terpene aroma components. Compared to traditional hot water extraction or organic solvent extraction, Citrus reticulata extract exhibits lower bitterness and better aroma, significantly enhancing its flavor. Specifically, the bitter substances naringin and neohesperidin in the Citrus reticulata extract are degraded, while some volatile substances such as aldehydes and alkenes undergo redox reactions, resulting in a stronger characteristic aroma of the Citrus reticulata extract. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: In this invention: naringinase, β-glucosidase, β-galactosidase, and cellulase can all be obtained through conventional commercial means and fall under the category of food additives. For example, naringinase, β-glucosidase, β-galactosidase, and cellulase can all be purchased from Shenzhen Lefu Biotechnology Co., Ltd.

[0022] Example 1: 1) Dry (moisture content ≤6%) Citrus reticulata powder is pulverized and passed through a 40-mesh sieve, then ultra-finely pulverized and passed through a 200-mesh sieve to obtain Citrus reticulata powder for later use.

[0023] 2) Weigh 5.0g of Citrus reticulata powder and mix it with distilled water at a mass ratio of 1:15. Let the powder absorb water and swell for 3 hours, and adjust the pH to 6.5 with 1N hydrochloric acid or 1N sodium hydroxide.

[0024] 3) Add 1 mL of 1 kU / mL naringinase and 1 mL of a complex hydrolytic enzyme solution containing 1 kU β-glucosidase, 1 kU β-galactosidase, and 1 kU cellulase to the solution obtained in step 2). Perform enzymatic hydrolysis for 2 hours in a 40°C constant temperature water bath with magnetic stirring. Filter the solution through a 1.2 μm membrane and pour the filtrate (approximately 50 mL) into a polyethylene plastic beaker (100 mL standard beaker), then place it in the sample chamber of the radio frequency cold plasma treatment device.

[0025] 4) Place the beaker containing the filtrate into the sample chamber of the radio frequency cold plasma treatment device (the total volume of the chamber is approximately 10L), then quickly seal the chamber. Adjust the vacuum pump to control the pressure (absolute pressure) inside the chamber to 0.3 bar, and then introduce argon gas (purity ≥99.99% pure argon gas) at a flow rate of 10 mL / min. Adjust the vertical distance between the dielectric barrier discharge radio frequency high-voltage electrode and the sample liquid surface to 9 cm. Use a dielectric barrier discharge structure, with an insulating dielectric barrier layer between the electrode and the discharge gap. Turn on the high-voltage AC power supply to perform glow discharge mode dielectric barrier cold plasma treatment. Set the discharge parameters as follows: voltage 60 kV, frequency 25 kHz, discharge power 450 W, and continuous isothermal cold plasma treatment for 20 min (radio frequency discharge 20 min).

[0026] 5) Turn off the radio frequency discharge and the vacuum pump, and wait for the radio frequency cold plasma treatment device to return to normal pressure; take out the sample, and you will get the processed Citrus reticulata extract sample #1.

[0027] Comparison 1: As a comparison, the extract of Citrus reticulata was prepared by boiling water extraction.

[0028] The main steps are as follows: Weigh 50g of Citrus reticulata powder, mix it with distilled water at a mass ratio of 1:15, allow the powder to absorb water and swell for 3 hours, then place it in an extraction tank, install a cooling water device, heat to boiling (about 99℃) and extract for 4 hours, stop extraction, cool to room temperature, and centrifuge the liquid (5000rpm, 10min). The supernatant is Citrus reticulata extract sample #2.

[0029] Experiment 1: Evaluation of bitterness and aroma.

[0030] The contents of naringin and neohesperidin were determined according to the analytical method of "Ultra-High Performance Synthesis Chromatography for the Determination of the Main Flavonoid Glycosides Naringin and Neohesperidin in External Application Formula for Gastroparesis" (Authors: Sun Yujie, Zhang Na, Cao Yijia, Qu Yuxia, Li Jianhao, Lin Hongying, Sun Yikun, Hu Kaiwen; *International Journal of Pharmaceutical Research*, April 2019, Vol. 46, No. 4, pp. 296-300). After diluting the samples with distilled water at a 1:1 mass ratio, the bitterness intensity was evaluated by seven sensory evaluators according to T / CSTM 01249-2024 *Guidelines for Sensory Analysis of Beverage and Alcoholic Beverages*, and the results are expressed as average values. The relative contents of characteristic volatile aroma components such as limonene were determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

[0031] The bitterness evaluation results (Table 1) show that the content of bitter components naringin and hesperidin in the Citrus reticulata extract prepared by this invention is much lower than that in samples extracted by traditional boiling water. Sensory evaluation results also indicate that the bitterness intensity of the Citrus reticulata extract prepared by this invention is much lower than that of samples extracted by traditional boiling water. Therefore, the technology of this invention can effectively reduce the bitterness of Citrus reticulata extract.

[0032] The relative contents of volatile aroma components in the samples were determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry. Based on the research results in the paper "Analysis and Quality Detection of Aroma Components in Citrus reticulata Extract Oil" (authors: Tan An, Chen Chusheng, Ma Yuyin, Wu Di, Kan Qixin, Song Mingyue; *Modern Food Science and Technology*, 2024, Vol. 40, No. 7, pp. 203-210), the contents of 15 characteristic aroma components of Citrus reticulata, including α-phenene, were compared. The results are shown in Table 2. Table 2 shows that the relative contents of these 15 characteristic aroma components in the extract prepared in this invention are significantly higher than those in the boiling water extract, especially the relative contents of olefins. This may be related to the use of glycosidases in the preparation process of this invention to release and volatilize some bound olefins, and the fact that the preparation at a lower temperature effectively reduces the oxidation of unsaturated olefins at high temperatures.

[0033] Table 1. Comparison of bitter substance content in the extracts of Citrus reticulata peel prepared by the present invention and by boiling water extraction method

[0034] Note: Different lowercase letters represent significant differences in the same row of data. p <0.05).

[0035] Table 2. Comparison of the contents of important aroma components in the Citrus reticulata extract prepared by the present invention and by boiling water extraction method.

[0036] Comparative Example 1, relative to Example 1: The use of "1 mL of a complex hydrolase solution containing 1 kU β-glucosidase, 1 kU β-galactosidase, and 1 kU cellulase" in step 3) was omitted, and the rest was the same as in Example 1.

[0037] Comparative Example 2 series, relative to Example 1: the "argon gas inlet flow rate 10 mL / min, radio frequency discharge 20 min" in Example 1 was changed to the following Table 3; the rest is the same as Example 1.

[0038] Comparative Example 3-1, relative to Example 1: the complex hydrolytic enzyme in step 3) was changed from "1kU β-glucosidase + 1kU β-galactosidase + 1kU cellulase solution 1mL" to "1.5kU β-glucosidase + 1.5kU β-galactosidase", and the rest was the same as in Example 1.

[0039] Comparative Example 3-2, relative to Example 1: the complex hydrolytic enzyme in step 3) was changed from "1kU β-glucosidase + 1kU β-galactosidase + 1kU cellulase solution 1mL" to "1.5kU β-glucosidase + 1.5kU cellulase", and the rest was the same as in Example 1.

[0040] Comparative Example 3-3, relative to Example 1: the complex hydrolytic enzyme in step 3) was changed from "1kU β-glucosidase + 1kU β-galactosidase + 1kU cellulase solution 1mL" to "1.5kU β-galactosidase + 1.5kU cellulase", and the rest was the same as in Example 1.

[0041] Table 3

[0042] The results of comparing all the above comparative examples with Example 1 according to "Experiment 1, Bitterness and Aroma Evaluation" are shown in Table 4 below.

[0043] Table 4

[0044] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling, characterized in that: After adding water to the tangerine peel powder, it is first enzymatically hydrolyzed, and then subjected to radio frequency cold plasma discharge treatment to obtain the tangerine peel extract.

2. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to claim 1, characterized in that... Includes the following steps: 1) Pulverize the dried Citrus reticulata peel to obtain Citrus reticulata peel powder; 2) Set the material-to-liquid mass ratio to 1:13~17; Add water to 5g of Citrus reticulata powder, and let the powder absorb water and swell for 3±0.5h, then adjust the pH to 6.5±0.

2. Alternatively, after adding water to 5g of Citrus reticulata powder, adjust the pH to 6.5±0.2, and then allow the Citrus reticulata powder to absorb water and swell for 3±0.5h. 3) Add 500~1000U of hydrolytic enzyme I and 1000~2000U of compound hydrolytic enzyme to the liquid obtained in step 2). Under magnetic stirring, hydrolyze at a constant temperature of 40±2°C for 2±0.1h, then filter and place the filtrate in a radio frequency cold plasma treatment device. 4) Control the pressure to 0.3±0.1 bar using a vacuum pump and introduce argon gas; The discharge parameters for controlling the radio frequency cold plasma treatment device are: voltage 60±3kV, frequency 25±1 kHz, discharge power 450±20 W, and treatment time 10~30min; The processed Citrus reticulata extract was obtained.

3. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to claim 2, characterized in that: After the processing time in step 4) is reached, turn off the radio frequency discharge and the vacuum pump, and wait for the radio frequency cold plasma treatment device to return to normal pressure; the processed tangerine peel extract is obtained.

4. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to claim 2 or 3, characterized in that... In step 4), the argon gas inlet flow rate is adjusted to 5~15 mL / min.

5. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to claim 4, characterized in that... In step 4): the distance between the radio frequency electrode and the liquid surface of the sample is adjusted to 9±0.5cm.

6. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to any one of claims 1 to 5, characterized in that... In step 3): Hydrolytic enzyme I is naringinase; The complex hydrolase consists of at least two of the following: β-glucosidase, β-galactosidase, and cellulase; the content of each enzyme in the complex hydrolase is 1000~2000U.

7. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to claim 6, characterized in that... In step 3), the complex hydrolase consists of 1 kU β-glucosidase, 1 kU β-galactosidase, and 1 kU cellulase.

8. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to any one of claims 1 to 7, characterized in that... In step 3): The filtration is performed through a 1.2μm filter membrane.

9. The method for enhancing the flavor and reducing the bitterness of Citrus reticulata extract using high-voltage radio frequency cold plasma-enzyme coupling according to any one of claims 1 to 8, characterized in that... In step 1), the citrus red powder is passed through a 200-mesh sieve.

Images