Method for enriching gamma-aminobutyric acid in potatoes and preparation and application of fermented product of gamma-aminobutyric acid

Abstract

The invention provides a method for enriching gamma-aminobutyric acid in potatoes and preparation and application of a fermented product of the gamma-aminobutyric acid, and belongs to the technical field of potato processing. The method comprises the following steps: preparing fresh-cut potatoes: dicing the potatoes for later use; enriching GABA (gamma-aminobutyric acid) through chlorogenic acid dipping treatment: dipping the diced potatoes into chlorogenic acid with the mass volume concentration of 8-12mg / L, taking out the diced potatoes, removing surface moisture, and refrigerating; carrying out anaerobic freezing treatment to enrich GAB; and performing vacuum anaerobic freezing treatment on the refrigerated diced potatoes. According to the invention, chlorogenic acid impregnation, anaerobic-freezing treatment and lactic acid bacteria fermentation are combined to form a set of efficient GABA enrichment process. According to the technology, the yield of GABA is increased, technical support is provided for high-value utilization of purple potatoes, and a new thought is provided for development of functional food rich in GABA.

Description

Technical Field

[0001] This invention relates to the field of potato processing technology, and in particular to a method for enriching γ-aminobutyric acid in potatoes and the preparation and application of its fermentation products. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid widely found in the living cells of organisms such as animals, plants, and microorganisms. In the human body, GABA is mainly converted from glutamate through the catalytic action of glutamate decarboxylase, with particularly high concentrations in the pancreas, second only to the central nervous system. As an inhibitory neurotransmitter, GABA reduces or inhibits the activity of cortical nerve activity, especially in the limbic system of the brain that controls emotions. Studies have shown that GABA has various physiological functions, including anti-anxiety, sleep aid, hormone regulation, and blood pressure reduction. Although the GABA content in plant tissues is usually low, plants can rapidly increase GABA accumulation when faced with adverse abiotic stresses such as hypoxia, heat shock, cold shock, mechanical damage, and ultrasound.

[0003] The main methods for producing GABA include plant enrichment and microbial fermentation. Microbial fermentation, in particular, offers advantages such as high safety, low cost, short cycle time, and lack of resource and environmental limitations, making it an ideal approach for large-scale GABA production. Common microbial strains used in GABA production via microbial fermentation include yeast, mold, lactic acid bacteria, and Escherichia coli. Lactic acid bacteria, in particular, are recognized as food-grade microorganisms with various physiological functions, including regulating intestinal flora, anti-oxidation, and enhancing immunity. The glutamate decarboxylase (GAD) activity of lactic acid bacteria is generally higher than that of other strains, giving it a significant advantage in GABA production for the food and pharmaceutical industries. Currently, GABA fermentation production primarily relies on starch and sugar raw materials; therefore, using inexpensive carbon sources can effectively reduce GABA production costs. The process involves synthesizing glutamate from carbohydrates and then decarboxylating the glutamate to produce GABA.

[0004] Potatoes, a crop widely used in food, vegetable, and feed industries, are highly nutritious and rich in GABA in their tubers, making them ideal as a raw material for producing GABA-rich functional foods. Studies have shown that abiotic stress methods such as alternating anaerobic-aerobic treatment, soaking, low-temperature treatment, and microbial fermentation can significantly increase the GABA content in potatoes. However, there are currently no studies, either domestically or internationally, on the effects of combined treatments such as soaking and anaerobic-freezing fermentation on GABA accumulation in potatoes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enriching γ-aminobutyric acid (GABA) in potatoes and the preparation and application of its fermentation products. By using chlorogenic acid to regulate the metabolic pathway of GABA in plants, the conversion of glutamate to GABA is promoted and the activity of GABA-degrading enzymes is inhibited, thereby effectively enriching GABA. At the same time, GABA is further enriched through anaerobic-freezing combined with lactic acid bacteria fermentation. This not only helps to improve the nutritional value of potatoes, but also provides a scientific basis for the development of functional foods.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for enriching γ-aminobutyric acid (GABA) in potatoes, comprising the following steps: Preparation of fresh-cut potatoes: Dice the potatoes and set aside; Enrichment of GABA by chlorogenic acid impregnation: Diced potatoes were impregnated in chlorogenic acid with a mass-volume concentration of 8-12 mg / L, removed and surface moisture was removed, and then refrigerated. GAB enrichment was achieved through anaerobic freezing; refrigerated potato cubes were subjected to vacuum anaerobic freezing.

[0007] Preferably, the potato is a purple potato; the potato is disinfected, dried and peeled before dicing; the ratio of potato cubes to chlorogenic acid in the liquid is 1g:1-3ml; and the soaking time is 8-12min.

[0008] Preferably, the refrigeration temperature is 3-5°C and the refrigeration time is 65-72 hours.

[0009] Preferably, the temperature of the vacuum anaerobic freezing is -15 to -20°C, and the time of the vacuum anaerobic freezing is 20-28 hours.

[0010] This invention also provides a method for preparing potato fermentation products enriched with γ-aminobutyric acid, comprising the following steps: The potato cubes that have undergone vacuum anaerobic freezing are thawed, mixed with water, homogenized, sterilized, and then lactic acid bacteria are added. The mixture is then fermented at a constant temperature to obtain a potato fermentation product enriched with γ-aminobutyric acid.

[0011] Preferably, the diced potatoes are purple diced potatoes; the ratio of diced potatoes to water is 1:5-7; the pulping time is 8-12 minutes; the primary homogenization pressure is 65-75 MPa, the secondary homogenization pressure is 95-105 MPa; the sterilization temperature is 85-95℃, and the sterilization time is 4-6 minutes.

[0012] Preferably, the amount of lactic acid bacteria added is 0.01-0.02g / 100ml, and the lactic acid bacteria is Lactobacillus plantarum.

[0013] Preferably, the temperature for constant temperature fermentation is 32-36℃, and the fermentation time is 12-16h.

[0014] The present invention also provides the application of the preparation method described above in the preparation of potato fermented beverages rich in γ-aminobutyric acid.

[0015] Preferably, the fermented potato beverage is a fermented purple potato beverage.

[0016] The beneficial effects of this invention compared to the prior art are as follows: This invention utilizes purple potatoes as a carbohydrate raw material. After chlorogenic acid impregnation and enrichment treatment, a purple potato beverage is fermented with lactic acid bacteria to enrich γ-aminobutyric acid (GABA). The effects of chlorogenic acid impregnation, anaerobic-freezing, and lactic acid bacteria fermentation on the GABA content in purple potatoes were investigated. Orthogonal experiments were used to optimize the GABA enrichment process conditions. It was found that a chlorogenic acid concentration of 10 mg / L, an impregnation time of 68 h, and a material-to-liquid ratio of 1:1 (g / mL) significantly increased GABA yield. Furthermore, this invention further enriched GABA by combining anaerobic-freezing and lactic acid bacteria fermentation. The optimal fermentation conditions were: lactic acid bacteria addition of 0.01 g / 100 mL, temperature of 35 °C, and fermentation time of 14 h. This indicates that the combination of physical and biological technologies can effectively increase the GABA content in purple potatoes.

[0017] The innovation of this invention lies in combining chlorogenic acid impregnation, anaerobic-freezing treatment, and lactic acid bacteria fermentation to form a highly efficient GABA enrichment process. This process not only increases GABA yield but also provides technical support for the high-value utilization of purple potatoes and offers new ideas for developing GABA-rich functional foods.

[0018] Furthermore, this invention, through GABA content determination, sensory evaluation, and antioxidant activity analysis, confirmed that the above-mentioned treatment significantly increased the GABA content of the purple potato fermented beverage. The GABA-rich purple potato fermented beverage not only possesses various physiological functions such as anti-anxiety, sleep aid, hormone regulation, and blood pressure reduction, but also exhibits excellent sensory quality and high antioxidant capacity. Its development is of great significance for promoting agricultural industrial restructuring, meeting consumer health needs, and enhancing product market competitiveness. This provides a theoretical basis for the deep processing of purple potatoes and the development of functional foods. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Technical route for developing fermented beverages rich in GABA from purple potatoes; Figure 2 This is the GABA standard curve; Figure 3 The effect of chlorogenic acid concentration on GABA enrichment in purple potatoes; Figure 4 The effect of chlorogenic acid soaking time on GABA enrichment in purple potatoes; Figure 5 The effect of the material-to-liquid ratio on GABA enrichment in purple potatoes; Figure 6 The effects of different storage methods on GABA enrichment in purple potatoes; Figure 7 The effect of lactic acid bacteria addition on the GABA content of purple potato fermented beverage; Figure 8 The effect of fermentation time on the GABA content of fermented purple potato beverages; Figure 9 The effect of fermentation temperature on the GABA content of fermented purple potato beverages; Figure 10 This is the IC50 curve. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] 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.

[0026] This invention provides a method for enriching γ-aminobutyric acid (GABA) in potatoes, comprising the following steps: Preparation of fresh-cut potatoes: Dice the potatoes and set aside; Enrichment of GABA by chlorogenic acid impregnation: Diced potatoes were impregnated in chlorogenic acid with a mass-volume concentration of 8-12 mg / L, removed and surface moisture was removed, and then refrigerated. GAB enrichment was achieved through anaerobic freezing; refrigerated potato cubes were subjected to vacuum anaerobic freezing.

[0027] In this invention, the potato is preferably a purple potato; the potato is preferably disinfected, dried, and peeled before dicing; disinfection is performed using 50ppm hypochlorous acid water, and the drying method is preferably natural air drying; the ratio of potato to chlorogenic acid is preferably 1g:1-3ml, more preferably 1g:1.5-2.5ml, and even more preferably 1g:2ml; the soaking time is preferably 8-12min, more preferably 10min; the principle of chlorogenic acid enriching GABA is mainly based on its regulatory effect on the GABA synthesis pathway in plants, promoting the conversion of glutamate to GABA: glutamate is an important substrate for GABA synthesis, and under the catalysis of glutamate decarboxylase (GAD), glutamate decomposes to form GABA; chlorogenic acid treatment can significantly increase the activity of GAD, thereby promoting the conversion of glutamate to GABA; GABA transaminase (GABA-T) is a key enzyme for decomposing GABA, and its reduced activity contributes to the accumulation of GABA; chlorogenic acid treatment can inhibit the activity of GABA-T. This reduces GABA decomposition; and GAD activity is affected by pH, chlorogenic acid treatment can improve GAD activity by providing a weakly acidic environment, thereby promoting GABA synthesis; however, if the concentration of chlorogenic acid is too high during the soaking process, it may inhibit the activity of microorganisms, affecting fermentation efficiency and product quality; after removal, it is preferable to place it on a clean gauze to air dry naturally; after soaking in chlorogenic acid for 10 minutes, the solution needs to be removed, short soaking time can avoid cell structure damage caused by overtreatment, ensure the integrity of plant tissue, and provide a good foundation for subsequent reactions; the mass-volume concentration of chlorogenic acid is preferably 10 mg / L; the two-step storage method is preferably: first, store at 3-5℃ for 65-72h, then place in (-15)-(-20)℃ for vacuum freezing storage for 20-28h; the storage temperature is preferably 4℃, the storage time is preferably 68-70h, and more preferably 69h; the vacuum freezing storage temperature is preferably -18℃, and the vacuum freezing storage time is preferably 22-26h, and more preferably 24h.

[0028] This invention also provides a method for preparing potato fermentation products enriched with γ-aminobutyric acid, comprising the following steps: The potato cubes that have undergone vacuum anaerobic freezing are thawed, mixed with water, homogenized, sterilized, and then lactic acid bacteria are added. The mixture is then fermented at a constant temperature to obtain a potato fermentation product enriched with γ-aminobutyric acid.

[0029] In this invention, the diced potatoes are preferably purple diced potatoes; the thawing method is preferably microwave thawing; the water is preferably purified water; the ratio of diced potatoes to water is preferably 1:5-7, more preferably 1:6; the pulping time is preferably 8-12 minutes, more preferably 10 minutes; after pulping, the mixture is preferably heated to 64-68°C, more preferably 65°C; the primary homogenization pressure is preferably 65-75 MPa, more preferably 68-72 MPa, and even more preferably 70 MPa; the secondary homogenization pressure is preferably 95-105 MPa, more preferably 98-102 MPa, and even more preferably 100 MPa; the homogenization time is preferably 10-20 minutes, more preferably 14-18 minutes, and even more preferably 15 minutes; the sterilization... The preferred method is water bath sterilization; the sterilization temperature is preferably 85-95℃, more preferably 88-92℃, and even more preferably 90℃; the sterilization time is preferably 4-6 min, more preferably 5 min; the amount of lactic acid bacteria added is preferably 0.01-0.02 g / 100 ml, more preferably 0.015 g / 100 ml; the lactic acid bacteria are preferably *Lactobacillus plantarum*; the constant temperature fermentation temperature is preferably 32-36℃, more preferably 34-35℃; the constant temperature fermentation time is preferably 12-16 h, more preferably 13-15 h, and even more preferably 14 h; after constant temperature fermentation, it is preferred to perform refrigeration treatment, the refrigeration temperature is preferably 3-5℃, more preferably 4℃; the refrigeration time is preferably 10-15 h, more preferably 12-14 h, and even more preferably 13 h.

[0030] In this invention, the lactic acid bacteria utilize their glutamate decarboxylase (GAD) system to convert glutamate into GABA, thereby significantly increasing the GABA content in the fermentation broth. Simultaneously, the decrease in pH during fermentation provides a suitable weakly acidic environment for GABA synthesis, further promoting GABA production. Lactic acid bacteria themselves possess various physiological functions, including regulating intestinal flora, enhancing immunity, and providing antioxidant effects. Their application in fermented beverages not only improves the nutritional value of the beverages but also enhances their health benefits, making them more readily accepted by consumers.

[0031] The present invention also provides the application of the preparation method described above in the preparation of potato fermented beverages rich in γ-aminobutyric acid.

[0032] In this invention, the fermented potato beverage is preferably a fermented purple potato beverage.

[0033] Specifically, this embodiment of the invention uses purple potato as an example to describe in detail the method for enriching γ-aminobutyric acid in potato.

[0034] Example 1 A method for enriching γ-aminobutyric acid (GABA) in potatoes includes the following steps: Disinfect and dry purple potatoes, then cut them into uniform small cubes. Immerse them in a 10 mg / L chlorogenic acid solution (98%, purchased from Macklin) at a material-to-liquid ratio of 1:1 (g / mL) for 10 minutes. Remove the purple potatoes and place them on a clean gauze. After the surface is free of excess moisture, place them in a plastic box with dimensions of 15 cm long, 10 cm wide, and 6 cm high. Store at 4℃ for 68 hours to obtain enriched purple potato cubes. Vacuum-sealed and then frozen at -18℃ for 24 hours to obtain vacuum-anaerobic frozen purple potato cubes.

[0035] Example 2 A method for enriching γ-aminobutyric acid, comprising the following steps: Disinfect, dry, and cut purple potatoes into uniform cubes. Immerse them in an 8 mg / L chlorogenic acid solution (98%, purchased from Macklin) at a material-to-liquid ratio of 1:2 (g / mL) for 12 minutes. Remove the purple potatoes and place them on a clean gauze. After the surface is free of excess moisture, place them in a plastic box with dimensions of 15 cm long, 10 cm wide, and 6 cm high. Store at 3°C ​​for 72 hours to obtain enriched purple potato cubes. Vacuum-sealed and frozen at -15°C for 28 hours to obtain vacuum-anaerobic frozen purple potato cubes.

[0036] Example 3 A method for enriching γ-aminobutyric acid, comprising the following steps: Disinfect, dry, and cut purple potatoes into uniform cubes. Immerse them in a 12 mg / L chlorogenic acid solution (98%, purchased from Macklin) at a material-to-liquid ratio of 1:3 (g / mL) for 8 minutes. Remove the purple potatoes and place them on a clean gauze. After the surface is free of excess moisture, place them in a plastic box with dimensions of 15 cm long, 10 cm wide, and 6 cm high. Store at 5°C for 65 hours to obtain enriched purple potato cubes. Vacuum-sealed and frozen at -20°C for 20 hours to obtain vacuum-anaerobic frozen purple potato cubes.

[0037] Example 4 After microwaving the diced purple potatoes from Example 1, which had undergone vacuum anaerobic freezing, they were mixed with purified water at a ratio of 1:6 and blended in a food processor for 10 minutes to ensure uniformity. The mixture was then heated to 65°C and homogenized in a homogenizer under the following conditions: first stage pressure 70 MPa, second stage pressure 100 MPa. After homogenization, the beverage was sterilized in a water bath at 90°C for 5 minutes to ensure the product's microbiological safety. The sterilized beverage was cooled to a suitable fermentation temperature, and 0.01 g / 100 ml of *Lactobacillus plantarum* was added. The mixture was then placed in a 35°C constant temperature incubator for 14 hours of fermentation. After fermentation, the mixture was cooled to room temperature to terminate the fermentation process. Aseptic filling was performed to ensure compliance with hygiene standards and to prevent microbial contamination. The product was then refrigerated at 4°C until its flavor and taste stabilized, yielding a fermented purple potato product enriched with γ-aminobutyric acid (GABA).

[0038] Example 5 After microwaving the diced purple potatoes from Example 2, which underwent vacuum anaerobic freezing, they were mixed with purified water at a ratio of 1:5 and blended in a food processor for 8 minutes to ensure uniformity. The mixture was then heated to 60°C and homogenized in a homogenizer under the following conditions: first stage pressure 65 MPa, second stage pressure 95 MPa. After homogenization, the beverage was sterilized in a water bath at 85°C for 6 minutes to ensure the product's microbiological safety. The sterilized beverage was cooled to a suitable fermentation temperature, and 0.01 g / 100 ml of lactic acid bacteria was added. It was then placed in a 32°C constant temperature incubator for 16 hours of fermentation. After fermentation, the beverage was cooled to room temperature to terminate the fermentation process. Aseptic filling was performed to ensure that the filling process met hygiene standards and to avoid microbial contamination. The product was then refrigerated at 3°C ​​until its flavor and taste stabilized, yielding a purple potato fermented product enriched with γ-aminobutyric acid (GABA).

[0039] Example 6 After microwaving the diced purple potatoes from Example 3, which underwent vacuum anaerobic freezing, they were mixed with purified water at a ratio of 1:7 and blended in a food processor for 12 minutes to ensure uniformity. The mixture was then heated to 70°C and homogenized in a homogenizer under the following conditions: first stage pressure 75 MPa, second stage pressure 105 MPa. After homogenization, the beverage was sterilized in a water bath at 95°C for 4 minutes to ensure the product's microbiological safety. The sterilized beverage was cooled to a suitable fermentation temperature, and 0.02 g / 100 ml of *Lactobacillus plantarum* (purchased from Wecon Probiotics (Suzhou) Co., Ltd.) was added. The mixture was then placed in a 36°C constant temperature incubator for 12 hours for fermentation. After fermentation, the mixture was cooled to room temperature to terminate the fermentation process. Aseptic filling was performed to ensure compliance with hygiene standards and to prevent microbial contamination. The product was then refrigerated at 5°C until its flavor and taste stabilized, yielding a purple potato fermented product enriched with γ-aminobutyric acid (GABA).

[0040] In this invention, all samples were measured three times. IBM SPSS Statistics 25.0 software was used to analyze the significance of the experimental data. P<0.05 was used to indicate that the difference reached the significance level, and P<0.01 was used to indicate that the effect was extremely significant. The results are expressed as x±SD.

[0041] The materials, reagents, instruments, and equipment involved in the following experimental examples are as follows: Fresh purple potatoes (provided by the laboratory); GABA-rich lactic acid bacteria (Lactobacillus fermentum); main reagents: GABA standard, Shanghai Yuanye Biotechnology Co., Ltd.; chlorogenic acid (98%), Macklin; o-phthalaldehyde ≥99% (HPLC), Shanghai Yuanye Biotechnology Co., Ltd.; sodium acetate solution, aladdin; hydrochloric acid, trichloroacetic acid, acetonitrile, and methanol solutions, Shanghai Yuanye Biotechnology Co., Ltd.; all chemicals used in this experiment were of analytical grade.

[0042] Main instruments and equipment: M1416R high-speed benchtop refrigerated centrifuge, Shenzhen Ruiwode Life Science & Technology Co., Ltd.; DL SB-5620 low-temperature coolant circulating pump, Zhengzhou Changsheng Experimental Instrument Co., Ltd.; H1650 benchtop high-speed centrifuge, Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co., Ltd.; DF-6CD magnetic stirring oil bath, Changzhou Yineng Experimental Instrument Factory; KQ-300DB CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; SHZ-D(Ⅲ) circulating water multi-purpose vacuum pump, Gongyi Honghua Instrument Equipment Industry and Trade Co., Ltd.; FE28 pH meter, Mettler Toledo Instruments (Shanghai) Co., Ltd.; ME204E / 02 electronic balance, Mettler Toledo Instruments (Shanghai) Co., Ltd.; vertical pressure steam sterilizer, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0043] Experimental Example 1: Investigation of a method for enriching GABA by chlorogenic acid impregnation of purple potatoes GABA Standard Curve Construction: Accurately pipette 1, 1.5, 3, 4.5, and 6 mL of 1 g / L GABA standard solution (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) into capped 10 mL centrifuge tubes, and add 9, 8.5, 7, 5.5, and 4 mL of distilled water respectively to obtain GABA standard solutions of different concentrations. High-performance liquid chromatography (HPLC) was used to process the standard GABA solutions of different concentrations. 100 μL of the standard was added to 500 μL of borate buffer (pH 10.4, 0.4 mol / L boric acid), mixed well, and then 100 μL of OPA solution was added. The mixture was reacted at room temperature for 2 min before being loaded onto the HPLC. A standard curve was plotted with absorbance on the x-axis and GABA concentration on the y-axis (e.g., ...). Figure 2 (As shown).

[0044] The equation of the standard curve is: y = 0.5474x + 654.24, R0 2 =0.9981, the absorbance value has a good linear relationship with the GABA mass concentration, and can be used as a standard curve for subsequent calculation of GABA content in the sample.

[0045] 1.1 Single-factor experiment on GABA enrichment in purple potatoes by chlorogenic acid impregnation The effects of chlorogenic acid concentration (0, 10, 30, 50, 70 mg / L), material-to-liquid ratio (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 g / mL), and reaction time (12, 24, 48, 72, 96 h) on the preparation of GABA from purple potato tubers were investigated using a single-factor experiment.

[0046] 1.1.1 Effect of chlorogenic acid concentration on the enrichment of GABA in purple potatoes After disinfecting, drying, and peeling purple potatoes, they were cut into uniform cubes. The cubes were then soaked in distilled water and chlorogenic acid solutions of concentrations of 10, 30, 50, and 70 mg / L for 10 minutes at a material-to-liquid ratio of 1:2 (g / mL). The filtrate was filtered and refrigerated at 4°C for 12 hours. Subsequently, 5g of the sample was accurately weighed, and the GABA content in the purple potatoes was determined using high-performance liquid chromatography (HPLC) to identify the optimal chlorogenic acid soaking concentration. The results are as follows: Figure 3 As shown.

[0047] The specific operating steps of high performance liquid chromatography are as follows: (1) Sample derivatization Accurately weigh 5g of sample, grind it, and place it in a 50mL centrifuge tube. Add 15mL of 0.1mol / L hydrochloric acid solution, mix well, sonicate for 30min, then heat in a 50℃ water bath for 30min, centrifuge at 8000r / min for 20min, and collect the supernatant. Take 100μL of the supernatant and mix it with 900μL of 20.0% trichloroacetic acid. After mixing well, centrifuge at 8000r / min for 20min, and filter the supernatant through a 0.45μm filter. Mix the filtered supernatant with o-phthalaldehyde solution (OPA, ≥99% (HPLC), purchased from Shanghai Yuanye Biotechnology Co., Ltd.) at a 1:1 ratio. Mix the above mixture thoroughly at room temperature and in the dark, and carry out the derivatization reaction. Calculate the GABA content of the sample using the plotted standard curve.

[0048] (2) HPLC analysis Mobile phase: Mobile phase A was sodium acetate solution; mobile phase B was sodium acetate-acetonitrile-methanol solution (1:2:2, V / V); chromatographic elution program: 0-24 min, 60.0% A; 24-30 min, 0% A; 30.1-35 min, 60% A; chromatographic column: C18 column (250 nm × 4.6 nm, 5 μm); detection wavelength: 338 nm; column oven temperature: 40℃; injection volume: 10 μL. Sodium acetate solution was purchased from Aladdin; hydrochloric acid, trichloroacetic acid, acetonitrile, and methanol solution were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; all chemicals used in this experiment were of analytical grade.

[0049] Depend on Figure 3 It can be seen that the production of GABA first increases and then decreases, with the highest GABA production achieved under treatment with 10 mg / L chlorogenic acid solution. Therefore, the chlorogenic acid concentration for subsequent optimization experiments was 10 mg / L.

[0050] 1.1.2 Effect of reaction time on the enrichment of GABA content in purple potatoes Purple potatoes were treated according to the method described in 1.1.1, and the GABA content in the purple potatoes was determined to identify the optimal soaking time. The concentration of the chlorogenic acid solution was set at 10 mg / L, and the refrigeration times were set at 12, 24, 48, 72, and 96 hours, respectively. The results are as follows: Figure 4 As shown.

[0051] Depend on Figure 4 It can be seen that the amount of GABA produced first increases and then decreases, reaching its highest level at an impregnation time of 72 hours. Therefore, the subsequent optimization experiment determined the impregnation time of chlorogenic acid to be 72 hours.

[0052] 1.1.3 Effect of the material-to-liquid ratio on the enrichment of GABA content in purple potatoes Purple potatoes were treated according to the method described in 1.1.1, and the GABA content in the purple potatoes was determined to identify the optimal soaking solution-to-solution ratio. The concentration of chlorogenic acid solution was set at 10 mg / L, the refrigeration time was set at 72 h, and the solution-to-solution ratios were set at 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5 (g / mL), respectively. The results are as follows: Figure 5 As shown.

[0053] Depend on Figure 5 It can be seen that the production of GABA first increases and then decreases, reaching its highest level when the material-to-liquid ratio is 1:1. Therefore, the material-to-liquid ratio for subsequent optimization experiments is 1:1.

[0054] 1.2 Orthogonal Experiment for GABA Enrichment in Purple Potatoes Impregnated with Chlorogenic Acid Based on the results of the single-factor experiment, the chlorogenic acid concentration was determined to be 10 mg / L, the reaction time was determined to be 72 h, the material-to-liquid ratio was determined to be 1:1 (g / mL), and L9(3) was used. 3 Orthogonal experiments were conducted to further optimize three factors: chlorogenic acid concentration, soaking time, and material-to-liquid ratio. Using the GABA content of purple potatoes as the standard, the optimal enrichment conditions were screened. The orthogonal experimental factor levels for chlorogenic acid enrichment of GABA are shown in Table 1. Purple potatoes were treated according to the method described in 1.1.1, and the GABA content in the purple potatoes was determined. The results are shown in Tables 2 and 3.

[0055] Table 1. Factor Levels in the Orthogonal Experiment for GABA Enrichment of Chlorogenic Acid

[0056] Table 2 Results of Orthogonal Experiments

[0057] Table 3. Analysis of Variance for Orthogonal Experiments

[0058] Note: * indicates a significant effect (P < 0.05), ** indicates a highly significant effect (P < 0.01). As shown in Tables 2 and 3, the range values ​​(r) of the three experimental factors are as follows: r B >r A >r C The reaction time had the greatest impact on GABA yield, followed by chlorogenic acid concentration and the solid-liquid ratio. Analysis of variance showed that chlorogenic acid concentration, soaking time, and solid-liquid ratio all had significant effects on GABA yield (P<0.05). The GABA content results obtained from the combinations of various factor levels showed that A2B1C2 was the optimal combination, meaning that under the reaction conditions of a chlorogenic acid concentration of 10 mg / L, a soaking time of 68 h, and a solid-liquid ratio of 1:1 (g / mL), the highest GABA production was achieved.

[0059] 1.3 Stability Experiment To verify the optimized process conditions, the potatoes were soaked in 10 mg / L chlorogenic acid solution for 10 min at a material-to-liquid ratio of 1 g:1 mL. After filtration, the filtrate was refrigerated at 4°C for 68 h, and the reaction was repeated three times. The GABA content in purple potatoes was determined according to the method in 1.1.1.

[0060] Repeated experiments showed that the average content of GABA generated in the three measurements was 133.08±2.75 mg / L, which was similar to the results of the orthogonal experiment, indicating that the optimized process conditions were stable and reliable.

[0061] 1.4 Anaerobic-freezing enrichment experiment of GABA Potato cubes enriched with chlorogenic acid under optimal conditions as described in 1.5 were vacuum-sealed and then placed at room temperature (15℃), refrigerated (4℃), and frozen (-18℃) for 24 hours to induce GABA enrichment under low-temperature stress. After removal, the potatoes were thawed by microwave heating, and their GABA content was determined according to the method in 1.1.1. The effects of different storage methods on GABA enrichment in purple potatoes were compared. The results are as follows: Figure 6 As shown.

[0062] Depend on Figure 6 It was found that the GABA content was highest in purple potato cubes stored anaerobic-frozen for 24 hours. This indicates that anaerobic-freezing is a highly efficient enrichment method. Therefore, anaerobic-freezing storage was determined to be the optimal storage method for purple potatoes.

[0063] Experimental Example 2: Exploration of the Fermentation Process of Purple Potato Beverage by Lactic Acid Bacteria 2.1 Single-factor experiment on lactic acid bacteria fermentation of purple potato beverage Using GABA-rich lactic acid bacteria and chlorogenic acid-enriched purple potato cubes as raw materials, the fermentation process was optimized using fermentation time (6, 12, 24, 48 h), compound bacterial powder addition (0.01, 0.02, 0.03, 0.04 g / 100 mL), and temperature (30, 35, 40, 45 °C) as evaluation indicators. The GABA content of the fermentation broth was also used as an evaluation indicator for process optimization.

[0064] 2.1.1 Effect of Lactic Acid Bacteria Addition Amount on the Accumulation of GABA in Purple Potato Beverage The purple potato cubes obtained in Example 1, after vacuum anaerobic freezing, were thawed using a microwave. Water was added at a material-to-liquid ratio of 1:6, and after homogenization, 0.01, 0.02, 0.03, and 0.04 g / 100 mL of lactic acid bacteria powder were added respectively. Fermentation was carried out at 30°C for 6 hours. Then, 5 mL of sample was accurately weighed, and the GABA content in the sample was determined according to the method in 1.1.1 to determine the optimal amount of lactic acid bacteria added. The results are as follows... Figure 7 As shown.

[0065] Depend on Figure 7 It can be seen that the production of GABA first increases and then decreases, reaching its highest level when the amount of lactic acid bacteria added is 0.02 g / 100 mL. Therefore, the amount of lactic acid bacteria added in subsequent optimization experiments was 0.02 g / 100 mL.

[0066] 2.1.2 Effect of fermentation time on the enrichment of GABA content in purple potato beverages Frozen purple potato cubes were processed according to the method described in section 2.1.1, and the GABA content in the samples was determined to establish the optimal lactic acid bacteria fermentation time. The amount of lactic acid bacteria powder added was set at 0.02 g / 100 mL, and the fermentation time was set at 6, 12, 24, and 48 h. The results are as follows: Figure 8 As shown.

[0067] Depend on Figure 8 It can be seen that the production of GABA first increases and then decreases, reaching its highest level at a fermentation time of 12 hours. Therefore, the subsequent optimization experiment determined the lactic acid bacteria fermentation time to be 12 hours.

[0068] 2.1.3 Effect of fermentation temperature on the enrichment of GABA content in purple potato beverages Frozen purple potato cubes were processed according to the method described in section 2.1.1, and the GABA content in the samples was determined to establish the optimal fermentation temperature for lactic acid bacteria. The dosage of lactic acid bacteria powder was set at 0.02 g / 100 mL, the fermentation time at 12 h, and the fermentation temperatures at 30, 35, 40, and 45 °C. The results are as follows: Figure 9 As shown.

[0069] Depend on Figure 9 It can be seen that the production of GABA first increases and then decreases, reaching its highest level at a fermentation temperature of 35℃. Therefore, the subsequent optimization experiment determined the fermentation temperature of the lactic acid bacteria to be 35℃.

[0070] 2.2 Orthogonal Experiment of Lactic Acid Bacteria Fermented Purple Potato Beverage Based on the single-factor experimental results in section 2.1, the optimal addition amount of lactic acid bacteria was determined to be 0.02 g / 100 mL, the fermentation time to be 12 h, and the fermentation temperature to be 35 °C. Furthermore, L9(3) was used... 3 An orthogonal experiment was conducted to optimize three factors: the amount of lactic acid bacteria added, fermentation time, and fermentation temperature, to investigate the effect on GABA in purple potato juice. The factor levels of the orthogonal experiment are shown in Table 4. Frozen purple potato cubes were treated according to the method in 2.1.1, and the GABA content in the samples was determined. The results are shown in Tables 5 and 6.

[0071] Table 4. Factor levels in the orthogonal experiment of lactic acid bacteria fermented purple potato beverage

[0072] Table 5. Results of the orthogonal experiment

[0073] Table 6. Analysis of Variance for Orthogonal Experiments

[0074] Note: * indicates a significant effect (P < 0.05), ** indicates a highly significant effect (P < 0.01). As shown in Tables 5 and 6, the ranges (r) of the three experimental factors are as follows: C >r B >r A Fermentation time had the greatest impact on GABA yield, followed by fermentation temperature and the amount of lactic acid bacteria added. Analysis of variance showed that the amount of lactic acid bacteria added and fermentation time had significant effects on GABA yield (P<0.05), while fermentation time had a highly significant effect on GABA yield (P<0.01). The GABA content results obtained from the combinations of factor levels showed that A1B2C3 was the optimal combination, meaning that under the reaction conditions of 0.01 g / 100 mL of lactic acid bacteria, a fermentation temperature of 35℃, and a fermentation time of 14 h, the highest GABA yield was achieved.

[0075] 2.3 Stability Experiment To verify the optimized process conditions, the amount of lactic acid bacteria added was set at 0.01 g / 100 mL, the fermentation temperature was set at 35 °C, and the fermentation time was set at 14 h. Frozen purple potato cubes were treated according to the method in 2.1.1 and the GABA content in the samples was determined.

[0076] The results showed that the average content of GABA generated in the three repeated experiments was 243.07±3.43 mg / L, which was similar to the results of the orthogonal experiment, indicating that the optimized process conditions were stable and reliable.

[0077] 2.4 Determination of antioxidant activity of fermented purple potato beverage The optimal conditions for fermented purple potato beverage samples were lyophilized. Subsequently, 0%, 1%, 5%, 10%, and 15% lyophilized powder were added during the reconstitution stage. The antioxidant activity of the fermented beverages at different concentrations was tested using a DPPH free radical scavenging assay kit (microplate method). The samples were mixed at a ratio of 1:9 with nitrogen free radical extract, centrifuged at 10,000 rpm for 10 min, and the supernatant was kept at 4℃. The microplate reader was preheated for at least 30 min, the wavelength was adjusted to 517 nm, and zeroed with anhydrous ethanol. A dose-response curve was established using a four-parameter nonlinear regression model, with the mathematical model: y = 11732.47 + (-0.11 - 11732.47) / [1 + (x / 4.56)0.19] (R 2 =0.997), and the calculated IC50 value is 4.56% when the clearance rate y = 50%. The results are as follows: Figure 10 As shown.

[0078] The calculation formula is as follows: Formula I; In Formula I: A0 - absorbance value of blank tube; A1 - absorbance value of sample test tube; A2 - absorbance value of sample control tube.

[0079] Depend on Figure 10 It can be seen that when the concentration of purple potato beverage reaches 4.56% (w / v), its scavenging effect on DPPH free radicals can reach 50% of the maximum effect, indicating that it has excellent antioxidant capacity.

[0080] 2.5 Physicochemical Indicators of Purple Potato Fermented Beverage The lightness (L*), red-green (a*), yellow-blue (b*), chromaticity (C*), and chromaticity angle (h°*) of the purple potato fermented beverage were measured using a colorimeter. The pH of the fermented beverage was tested using a pH meter, and the viscosity was tested using a rotational viscometer. Each sample was measured three times, and the average value was taken as the final result. The results are shown in Table 7.

[0081] Table 7 Physicochemical Indicators of Purple Potato Fermentation Beverage

[0082] The pH of the fermented purple potato beverage, measured under the optimal processing formula, was 5.73, and the viscosity was 7.86 mPa·s, classifying it as a low-viscosity beverage. It has a light and thin texture, without a thick or sticky feel upon entry. The brightness (L) is 21.24, indicating a darker color. The red-green axis (a) is -5.59, a negative value indicating a greenish tint. The yellow-blue axis (b) is -2.14, a negative value indicating a blueish tint. The chromaticity (C) is 6.77, indicating moderate saturation. The hue angle (h°) is 198.44°, indicating a color between blue and green. The beverage's color can be described as a dark blue-green with moderate saturation. The color is uniform, with no significant color difference or fading.

[0083] 2.6 Fuzzy Mathematical Sensory Evaluation of Purple Potato Fermented Beverage Fermented purple potato products were prepared according to the method in Example 4. Referring to GB / T 31121-2014 Fruit and Vegetable Juices and Beverages, a sensory evaluation team of 10 students trained in sensory evaluation conducted a sensory evaluation on five groups of fermented purple potato beverages (samples 1, 2, 3, 4, and 5) prepared with different material-to-liquid ratios (1:3, 1:4, 1:5, 1:6, and 1:7). The evaluation assessed four aspects: appearance, color, flavor, and taste. Each sample was scored as excellent, average, or poor. The evaluation criteria and weight distribution are shown in Tables 8 and 9, respectively. The corresponding distribution of votes for each grade is shown in Table 10. The evaluation results are shown in Table 11.

[0084] Table 8 Sensory ratings of purple potato fermented beverages

[0085] Table 9. Weight distribution of quality factors in purple potato fermented beverages

[0086] Convert the votes for each quality factor level of each sample in Table 10 into the approval ratio, and combine this with the evaluation results of all factors for each sample to obtain the fuzzy matrix Aj. The fuzzy matrix for sample j is: Aj= Formula II; Where: j=1, 2, 3, 4 are sample numbers, i=1, 2, 3, 4 are quality evaluation factors, and ri1, ri2, ri3 are the approval ratios of the votes obtained for each evaluation level of the i-th evaluation factor.

[0087] Table 10 Distribution of Sensory Evaluation Votes

[0088] Note: v1: Excellent; v2: Average; v3: Poor.

[0089] The fuzzy matrix is: Aj= A1= A2=

[0090] A3= A4= A5=

[0091] According to the principle of fuzzy matrix transformation: R = K × A, the evaluation result for sample j is Rj = K × Aj. Taking the first group of samples as an example, the comprehensive evaluation result is: R1=K×A1=(0.29, 0.15, 0.25, 0.31)× =(0.23,0.125,0.7225), Equation III; R2=K×A2=(0.29, 0.15, 0.25, 0.31)× =(0.0725,0.3785,0.4275), Equation IV; R3=K×A3=(0.29, 0.15, 0.25, 0.31)× =(0.416,0.3,0.0925), Equation V; R4=K×A4=(0.29, 0.15, 0.25, 0.31)× =(0.6575,0.2275,0.029), Equation VI; R5=K×A5=(0.29, 0.15, 0.25, 0.31)× =(0.4255,0.2335,0.1365), Equation VII; After obtaining the above results, assign values ​​to the evaluation criteria (v1=9, v2=6, v3=3), and then multiply them by R1. Y1=R1× =4.9875, Equation VIII; Similarly, the sensory scores for other samples were obtained, as shown in Table 11.

[0092] Table 11 Overall ranking results of sensory evaluation of purple potato fermented beverages

[0093] As shown in Table 11, when the ratio of purple potatoes to water is 1:6, the fermented beverage has the best sensory quality and is the most popular.

[0094] As can be seen from the above embodiments, the present invention provides a method for enriching γ-aminobutyric acid in potatoes and the preparation and application of its fermentation products. The present invention not only effectively enriches GABA by using chlorogenic acid to regulate the metabolic pathway of GABA in plants, promoting the conversion of glutamate to GABA, and inhibiting the activity of GABA-degrading enzymes, but also further enriches GABA through anaerobic-freezing combined with lactic acid bacteria fermentation treatment.

[0095] 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 enriching γ-aminobutyric acid (GABA) in potatoes, characterized in that, Includes the following steps: Preparation of fresh-cut potatoes: Dice the potatoes and set aside; Enrichment of GABA by chlorogenic acid impregnation: Diced potatoes were impregnated in chlorogenic acid with a mass-volume concentration of 8-12 mg / L, removed and surface moisture was removed, and then refrigerated. GAB enrichment was achieved through anaerobic freezing; refrigerated potato cubes were subjected to vacuum anaerobic freezing.

2. The method according to claim 1, characterized in that, The potatoes are purple potatoes; the potatoes are disinfected, dried and peeled before dicing; the ratio of potato cubes to chlorogenic acid in the liquid is 1g:1-3ml; the soaking time is 8-12min.

3. The method according to claim 1, characterized in that, The refrigeration temperature is 3-5℃, and the refrigeration time is 65-72h.

4. The method according to claim 1, characterized in that, The temperature of the vacuum anaerobic freezing is -15 to -20°C, and the time of the vacuum anaerobic freezing is 20-28 hours.

5. A method for preparing a potato fermentation product enriched with γ-aminobutyric acid, characterized in that, Includes the following steps: Thaw the potato cubes that have undergone vacuum anaerobic freezing as described in any one of claims 1-4, add water to make a pulp, homogenize, sterilize, add lactic acid bacteria, and ferment at a constant temperature to obtain a potato fermentation product enriched with γ-aminobutyric acid.

6. The method according to claim 5, characterized in that, The diced potatoes are purple diced potatoes; the ratio of diced potatoes to water is 1:5-7; the pulping time is 8-12 minutes; the primary homogenization pressure is 65-75 MPa, the secondary homogenization pressure is 95-105 MPa; the sterilization temperature is 85-95℃, and the sterilization time is 4-6 minutes.

7. The method according to claim 5, characterized in that, The amount of lactic acid bacteria added is 0.01-0.02g / 100ml, and the lactic acid bacteria is Lactobacillus plantarum.

8. The method according to claim 5, characterized in that, The temperature for constant temperature fermentation is 32-36℃, and the fermentation time is 12-16h.

9. The application of the preparation method according to any one of claims 5-8 in the preparation of potato fermented beverages rich in γ-aminobutyric acid.

10. The application according to claim 9, characterized in that, The fermented potato beverage is a fermented purple potato beverage.

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