Method for extracting natural antioxidant from cyperus esculentus shell fermentation product by using subcritical water

Abstract

The invention discloses a method for extracting a natural antioxidant from a cyperus esculentus shell fermentation product by using subcritical water, and belongs to the technical field of plant antioxidant extraction. The preparation method comprises the following steps: fermenting cyperus esculentus shell powder to obtain fermentation liquor; performing subcritical water extraction on the fermentation liquor to obtain an extracting solution; and filtering, purifying and freeze-drying the extracting solution to obtain the natural antioxidant. The plant cell walls can be selectively and mildly degraded and deconstructed by utilizing rich enzyme systems secreted by specific microorganisms in the growth and metabolism process. The biological pretreatment not only can destroy the physical barrier of the cell wall, but also can release active substances in a combined state in the cell wall, so that the active substances are converted into a free state, and the dissolution efficiency and the yield of subsequent extraction are greatly improved. Subcritical water extraction has the outstanding advantages of high extraction efficiency, high speed, no need or great reduction of the use amount of organic solvents, environmental friendliness and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant antioxidant extraction technology, specifically relating to a method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water. Background Technology

[0002] Tiger nuts (Cyperus esculentus) are a nutrient-rich tuberous plant, and their cultivation and utilization are receiving increasing attention globally. During tiger nut oil processing, the outer shell of the tuber is generated in large quantities as a major byproduct. Currently, aside from a small amount used as animal feed or discarded, the vast majority of these shells are not effectively utilized, resulting in resource waste and environmental pressure.

[0003] However, recent studies have found that tiger nut shells are not useless waste; their tissues contain abundant natural active ingredients, including polyphenols (such as phenolic acids and flavanols), flavonoids (such as flavonoid glycosides and flavonoid aglycones), and glycosides. These components have been proven to have significant antioxidant, antibacterial, and anti-inflammatory biological activities, indicating that tiger nut shells have great development potential in functional foods, health products, and natural cosmetics, especially as a natural alternative to synthetic antioxidants (such as BHT and BHA).

[0004] To realize the aforementioned value, the efficient and environmentally friendly extraction of active ingredients from plant matrices is a crucial first step. Currently, the extraction of antioxidant components from plants largely relies on traditional methods, such as hot water extraction and organic solvent methods (e.g., methanol, ethanol, acetone).

[0005] Thermal extraction: Prolonged high-temperature treatment can easily lead to the oxidation, degradation or isomerization of heat-sensitive polyphenols and flavonoids, thereby significantly reducing their biological activity.

[0006] Organic solvent extraction: Although widely used, it has problems such as solvent residue, environmental pollution, high energy consumption and potential health risks, which does not conform to the development concept of "green chemistry".

[0007] Bottleneck in extraction efficiency: More importantly, tiger nut shells have a dense plant cell wall structure (mainly composed of cellulose, hemicellulose and lignin). This natural physical barrier severely hinders the penetration of extraction solvent into the cell and the dissolution of intracellular active ingredients outward, resulting in low extraction efficiency and unsatisfactory yield.

[0008] Therefore, developing a combined pretreatment and extraction technology that can overcome the cell wall barrier, is an urgent need to realize the high-value-added resource utilization of tiger nut shells. Summary of the Invention

[0009] In view of this, the purpose of this invention is to provide a method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water. The process of this invention aims to create favorable conditions for subsequent subcritical water extraction through the "bio-wall disruption" effect of microorganisms; and then utilize the green and efficient "physical extraction" capabilities of subcritical water to maximize the recovery of polar antioxidant components from tiger nut shells.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water, comprising the following preparation steps: S1. Ferment the tiger nut shell powder to obtain a fermentation liquid; S2. The fermentation broth is extracted using subcritical water extraction to obtain an extract; S3. The extract is filtered, purified, and freeze-dried to obtain a natural antioxidant.

[0011] Preferably, the tiger nut shell powder in S1 has a mesh size of 40-80.

[0012] Preferably, the fermentation step in S1 is as follows: tiger nut shell powder and phosphate buffer are mixed at a mass ratio of 1:10, heated and soaked at 60-65℃ for 1.5-2.5 hours to kill miscellaneous bacteria; the temperature is lowered to 25-35℃ and then 1%wt of mixed bacterial strain is inoculated, and the fermentation is carried out at 200 rpm for 2-3 days under aerobic conditions.

[0013] Preferably, the mixed strain is obtained by mixing Bacillus subtilis and Trichoderma reesei at a live count ratio of 1:1.

[0014] Preferably, the parameters for subcritical water extraction in S2 are: Temperature 120–180 ℃; Pressure: 0.19–1.01 MPa; Duration: 20–240 minutes; Stirring speed 60 rpm.

[0015] Preferably, the purification step in S3 is as follows: the extract is mixed with ethanol to a system ethanol concentration of 60-80% v / v; then it is refrigerated at 4°C and allowed to stand for 12-24 hours; then the precipitate is removed by centrifugation to obtain the supernatant. The supernatant was subjected to resin adsorption, elution, concentration, and freeze-drying.

[0016] Preferably, the resin is AB-8, NKA-9, D101, or HPD-100.

[0017] It contains at least the following beneficial technical effects: This invention utilizes the abundant enzyme systems secreted by specific microorganisms during their growth and metabolism to selectively and gently degrade and deconstruct plant cell walls. This biological pretreatment not only disrupts the physical barrier of the cell wall but also releases bound active substances from the cell wall, converting them into a free state, thereby significantly improving the dissolution efficiency and yield of subsequent extractions. Subcritical water extraction has outstanding advantages such as high extraction efficiency, fast speed, no or significantly reduced use of organic solvents, and environmental friendliness.

[0018] The process of this invention aims to create favorable conditions for subsequent subcritical water extraction through the "bio-wall disruption" effect of microorganisms; then, by utilizing the green and efficient "physical extraction" capabilities of subcritical water, the polar antioxidant components in tiger nut shells can be recovered to the maximum extent. This not only effectively overcomes the shortcomings of traditional extraction methods but also fully aligns with the direction of sustainable development, providing a promising technical approach for the full-value and high-value utilization of tiger nut processing byproducts. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0025] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0026] Example 1 A method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water includes the following preparation steps: S1. Mix 60-mesh tiger nut shell powder with phosphate buffer at a mass ratio of 1:10 and heat at 65°C for 2 hours to kill contaminants; then lower the temperature to 30°C and inoculate with 1%wt of a mixed bacterial strain (obtained by mixing Bacillus subtilis and Trichoderma reesei at a viable count of 1:1), maintain a rotation speed of 200 rpm, and ferment aerobically for 2.5 days to obtain the fermentation broth; S2. The fermentation broth is added to a sealed extraction vessel and extracted by subcritical water extraction to obtain the extract; the parameters for subcritical water extraction are: Temperature 150℃; Pressure 0.47 MPa; Time: 90 minutes; Stirring speed 60 rpm.

[0027] S3. Filter the extract, mix the extract with ethanol to a system ethanol concentration of 70% v / v; then refrigerate at 4°C for 18 hours, and then centrifuge to remove the precipitate to obtain the supernatant; The supernatant was adsorbed, eluted, concentrated, and freeze-dried using AB-8 resin to obtain a natural antioxidant.

[0028] Example 2 A method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water includes the following preparation steps: S1. Mix 80-mesh tiger nut shell powder with phosphate buffer at a mass ratio of 1:10 and heat at 65°C for 2.5 hours to kill contaminants; then lower the temperature to 35°C and inoculate with 1%wt of a mixed bacterial strain (obtained by mixing Bacillus subtilis and Trichoderma reesei at a viable count of 1:1), maintain a rotation speed of 200 rpm, and ferment aerobically for 3 days to obtain the fermentation broth; S2. The fermentation broth is added to a sealed extraction vessel and extracted by subcritical water extraction to obtain the extract; the parameters for subcritical water extraction are: Temperature 180 ℃; Pressure 1.01 MPa; Time: 20 minutes; Stirring speed 60 rpm.

[0029] S3. Filter the extract, mix the extract with ethanol to a system ethanol concentration of 80% v / v; then refrigerate at 4°C for 24 hours, and then centrifuge to remove the precipitate to obtain the supernatant; The supernatant was adsorbed, eluted, concentrated, and freeze-dried using NKA-9 resin to obtain a natural antioxidant.

[0030] Example 3 A method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water includes the following preparation steps: S1. Mix 40-mesh tiger nut shell powder with phosphate buffer at a mass ratio of 1:10 and heat at 60℃ for 1.5 hours to kill contaminants; then lower the temperature to 25℃ and inoculate with 1%wt of a mixed bacterial strain (obtained by mixing Bacillus subtilis and Trichoderma reesei at a viable count of 1:1), maintain a rotation speed of 200 rpm, and ferment aerobically for 2 days to obtain the fermentation broth; S2. The fermentation broth is added to a sealed extraction vessel and extracted by subcritical water extraction to obtain the extract; the parameters for subcritical water extraction are: Temperature 120℃; Pressure 0.19 MPa; Time: 240 minutes; Stirring speed 60 rpm.

[0031] S3. Filter the extract, mix the extract with ethanol to a system ethanol concentration of 60% v / v; then refrigerate at 4°C for 12 hours, and then centrifuge to remove the precipitate to obtain the supernatant; The supernatant was adsorbed, eluted, concentrated, and freeze-dried using HPD-100 resin to obtain a natural antioxidant.

[0032] Experimental Example 1 The product obtained in Example 1, as determined by the Folin-Ciocalteu method, had a total phenolic content of 85 mg GAE·g. - ¹(dry basis), DPPH free radical scavenging IC 50 0.35 mg·mL - ¹, The overall extraction rate on a dry basis is 5.5%.

[0033] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting natural antioxidants from tiger nut shell fermentation products using subcritical water, characterized in that, The preparation steps include the following: S1. Ferment the tiger nut shell powder to obtain a fermentation liquid; S2. The fermentation broth is extracted using subcritical water extraction to obtain an extract; S3. The extract is filtered, purified, and freeze-dried to obtain a natural antioxidant.

2. The method according to claim 1, characterized in that, The tiger nut shell powder in S1 has a mesh size of 40-80.

3. The method according to claim 1, characterized in that, The fermentation step in S1 is as follows: mix tiger nut shell powder with phosphate buffer at a mass ratio of 1:10, heat and soak at 60-65℃ for 1.5-2.5 hours to kill miscellaneous bacteria; lower the temperature to 25-35℃ and then inoculate with 1%wt of mixed bacterial strain, maintain a rotation speed of 200 rpm, and ferment aerobically for 2-3 days.

4. The method according to claim 3, characterized in that, The mixed strain was obtained by mixing Bacillus subtilis and Trichoderma reesei at a live count ratio of 1:

1.

5. The method according to claim 1, characterized in that, The parameters for subcritical water extraction in S2 are as follows: Temperature 120–180 ℃; Pressure 0.19 ~ 1.01 MPa; Duration: 20–240 minutes; Stirring speed 60 rpm.

6. The method according to claim 1, characterized in that, The purification step in S3 is as follows: the extract is mixed with ethanol to a system ethanol concentration of 60-80% v / v; then it is refrigerated at 4°C and allowed to stand for 12-24 hours; then the precipitate is removed by centrifugation to obtain the supernatant. The supernatant was subjected to resin adsorption, elution, concentration, and freeze-drying.

7. The method according to claim 6, characterized in that, The resin is AB-8, NKA-9, D101, or HPD-100.