A method for extracting purified gamma-aminobutyric acid from ice plant

Abstract

The present invention provides a method for extracting purified gamma-aminobutyric acid from ice plants, belonging to the technical field of gamma-aminobutyric acid extraction, and the method comprises the following steps: (1) anaerobic-aerobic alternating treatment of ice plants, crushing, soaking, and obtaining an ice plant pre-extract solution; (2) constant temperature extraction of the ice plant pre-extract solution, concentration, and obtaining a crude extract; (3) crude extract removal, sterilization, and obtaining a purified gamma-aminobutyric acid. The present invention uses ice plants as raw materials, adopts an anaerobic-aerobic alternating treatment, soaking first and then constant temperature extraction method for extraction, and obtains a purified gamma-aminobutyric acid with a content of 4.9 to 5.3 mg / g, no odor, white or light yellow loose powder crystals, and granules. The purified gamma-aminobutyric acid meets the requirements of production and processing, has good processing quality, and mouse experiments show that the purified gamma-aminobutyric acid prepared by the present invention has the effect of improving immune function and enhancing immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of gamma-aminobutyric acid extraction, and in particular relates to a method for extracting purified gamma-aminobutyric acid from ice plants. Background Art

[0002] Ice plant is a nutritious medicinal material, also known as ice grass, ice leaf day flower, ice flower, etc. Ice plant is cool in nature and has the effects of cooling and relieving heat, promoting body fluid and quenching thirst, relieving asthma, and stopping bleeding. It is rich in amino acids, carotene, potassium, sodium, flavonoids and a small amount of inositol, and has the effects of preventing eye diseases, preventing cardiovascular and cerebrovascular diseases, reducing inflammation and sterilizing, and inhibiting the growth of cancer cells.

[0003] The GABA (γ-aminobutyric acid) contained in ice plant is a natural non-protein amino acid. It is a substance that inhibits nerve transmission and is mostly found in animals. γ-aminobutyric acid can not only lower blood pressure, calm down, resist convulsions, improve liver and kidney function, and improve sleep quality, but also has anti-oxidation, enhances immune function, increases brain activity, accelerates ethanol metabolism, and improves exercise tolerance.

[0004] Currently, there is no method that can effectively extract GABA from ice plant. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for extracting purified γ-aminobutyric acid from ice plant. The method provided by the present invention can obtain white or light yellow loose powdery crystals and granules with a content of 4.9-5.3 mg / g of purified γ-aminobutyric acid that meets production requirements and can improve the immune function of mice.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting purified gamma-aminobutyric acid from ice plant, comprising the following steps:

[0008] (1) treating the ice plant in an anaerobic-aerobic alternating manner, crushing, and soaking to obtain an ice plant pre-extract solution;

[0009] (2) extracting the pre-extracted ice plant at a constant temperature, and concentrating the extract to obtain a crude extract;

[0010] (3) The crude extract is purified and sterilized to obtain purified γ-aminobutyric acid.

[0011] Preferably, the temperature of the anaerobic-aerobic alternating treatment in step (1) is 30-40° C., the number of the anaerobic-aerobic alternating treatment is 2-4 times, and the method of the anaerobic-aerobic alternating treatment is: placing the ice plant under anaerobic conditions for 2-3 hours, and then placing it under aerobic conditions for 1-2 hours.

[0012] Preferably, the powder in step (1) is sieved through a 50-60 mesh screen after being crushed.

[0013] Preferably, ethanol is used for soaking in step (1), the soaking temperature is 25-30° C., and the soaking time is 30-40 min.

[0014] Preferably, the volume fraction of the ethanol is 70-80%, and the volume mass ratio of the ethanol to the ice plant is 8-10 ml:1 g.

[0015] Preferably, the temperature of the constant temperature extraction in step (2) is 90-100° C., the time of the constant temperature extraction is 5-10 min / time, and the number of constant temperature extractions is 2-4 times.

[0016] Preferably, the concentration temperature in step (2) is 50-60° C., and the concentration time is 1-2 h.

[0017] Preferably, the method for removing impurities in step (3) is to mix the crude extract with ethanol, react for 1 to 2 hours, filter, and obtain a de-impurified product.

[0018] Preferably, the volume fraction of the ethanol is 90-95%, and the mass volume ratio of the crude extract to the ethanol is 1 g:2-3 ml.

[0019] Preferably, the sterilization temperature in step (3) is 90-110° C., and the sterilization time is 0.5-1 h.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention uses ice plant as a raw material for extracting γ-aminobutyric acid, and adopts an anaerobic-aerobic alternating treatment method, a soaking method and a constant temperature extraction method for extraction, to obtain a purified γ-aminobutyric acid with a content of 4.9-5.3 mg / g, a unique flavor of the product, no peculiar smell, and white or light yellow loose powdery crystals. The results of physical and chemical and sanitary inspections show that the purified γ-aminobutyric acid prepared by the present invention has good processing quality and can be used as a food additive in the future. At the same time, mouse experiments show that the purified γ-aminobutyric acid prepared by the present invention can improve the immunity of high-fat model mice and has the effect of improving immune function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The standard curve is prepared after measuring the γ-aminobutyric acid standard solution. DETAILED DESCRIPTION

[0023] The present invention provides a method for extracting purified gamma-aminobutyric acid from ice plant, comprising the following steps:

[0024] (1) treating the ice plant in an anaerobic-aerobic alternating manner, crushing, and soaking to obtain an ice plant pre-extract solution;

[0025] (2) extracting the pre-extracted ice plant at a constant temperature, and concentrating the extract to obtain a crude extract;

[0026] (3) The crude extract is purified and sterilized to obtain purified γ-aminobutyric acid.

[0027] In the present invention, the ice plant is treated alternately by anaerobic-aerobic treatment, crushed, and soaked to obtain the ice plant pre-extract solution.

[0028] In the present invention, the temperature of the anaerobic-aerobic alternating treatment is preferably 30-40°C, more preferably 32-38°C, and more preferably 34-36°C; the number of the anaerobic-aerobic alternating treatment is preferably 2-4 times, and more preferably 3 times; the method of the anaerobic-aerobic alternating treatment is preferably: placing the ice plant under anaerobic conditions for 2-3 hours, and then placing it under aerobic conditions for 1-2 hours, and more preferably placing the ice plant under anaerobic conditions for 2.5 hours, and then placing it under aerobic conditions for 1.5 hours; the anaerobic-aerobic alternating treatment can maintain the original color and aroma of the ice plant, reduce the browning degree of the ice plant, and the treatment method can increase the content of γ-aminobutyric acid obtained by extraction, thereby facilitating the improvement of the extraction efficiency of γ-aminobutyric acid. The ice plant is preferably a whole plant that is healthy and free of diseases and insect pests; after the crushing, it is preferably passed through a 50-60 mesh sieve; ethanol is preferably used for soaking, and the soaking temperature is preferably 25-30°C, more preferably 26-28°C, and further preferably 27°C; the soaking time is preferably 30-40min, more preferably 32-38min, and further preferably 34-36min; the volume fraction of the ethanol is preferably 70-80%, more preferably 72-78%, and further preferably 74-76%; the volume mass ratio of the ethanol to the ice plant is preferably 8-10ml:1g, and further preferably 9ml:1g.

[0029] In the present invention, the pre-extracted solution of Psoralea corylifolia is extracted at a constant temperature and concentrated to obtain a crude extract.

[0030] In the present invention, the temperature of the constant temperature extraction is preferably 90-100°C, more preferably 92-98°C, and more preferably 95°C; the time of the constant temperature extraction is preferably 5-10 min / time, more preferably 6-8 min / time, and more preferably 7 min / time; the number of constant temperature extractions is preferably 2-4 times, and more preferably 3 times; the constant temperature extraction can increase the permeability of the cell membrane and cell wall, making it easier for the solvent to enter the cell and increase the dissolution of γ-aminobutyric acid. The temperature of the concentration is preferably 50-60°C, more preferably 52-58°C, and more preferably 54-56°C; the time of the concentration is preferably 1-2h, and more preferably 1.5h.

[0031] In the present invention, the crude extract is purified and sterilized to obtain a purified γ-aminobutyric acid.

[0032] In the present invention, the method for removing impurities is preferably to mix the crude extract with ethanol, react for 1 to 2 hours, filter, and obtain a de-impurity product, and the reaction time is further preferably 1.5 hours; the volume fraction of the ethanol is preferably 90 to 95%, further preferably 92 to 94%, and further preferably 93%; the mass volume ratio of the crude extract to ethanol is preferably 1g:2 to 3ml, and further preferably 1g:2.5ml; the sterilization temperature is preferably 90 to 110°C, further preferably 95 to 105°C, and further preferably 100°C; the sterilization time is preferably 0.5 to 1h, further preferably 0.6 to 0.8h, and further preferably 0.7h; and after the sterilization, disinfection and packaging are preferably performed.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A method for extracting purified gamma-aminobutyric acid from ice plant, comprising the following steps:

[0036] (1) placing the ice plant at 30° C. under anaerobic conditions for 3 hours, and then placing it at 30° C. under aerobic conditions for 2 hours, repeating the above steps twice, crushing and passing through a 50-mesh sieve, adding the sieved ice plant to a 70% by volume ethanol solution at a volume mass ratio of ethanol to ice plant of 8 ml:1 g, and soaking for 30 minutes to obtain an ice plant pre-extract solution;

[0037] (2) extracting the pre-extract of ice plant at 100°C for 10 min / time, extracting at this temperature for 3 times, and concentrating at 50°C for 2 h to obtain a crude extract;

[0038] (3) The crude extract was mixed with 90% ethanol by volume at a mass volume ratio of 1 g:2 ml, reacted for 2 h, filtered, and sterilized at 100° C. for 1 h to obtain purified γ-aminobutyric acid.

[0039] Example 2

[0040] A method for extracting purified gamma-aminobutyric acid from ice plant, comprising the following steps:

[0041] (1) placing the ice plant at 40° C. under anaerobic conditions for 2 hours, and then placing it at 40° C. under aerobic conditions for 1 hour, repeating the above steps 4 times, crushing and passing through a 60-mesh sieve, adding the sieved ice plant to an 80% ethanol solution with a volume mass ratio of ethanol to ice plant of 10 ml:1 g, and soaking for 40 minutes to obtain an ice plant pre-extract solution;

[0042] (2) extracting the pre-extract of ice plant at 90°C for 5 min each time, extracting at this temperature for 4 times, and concentrating at 60°C for 1 h to obtain a crude extract;

[0043] (3) The crude extract was mixed with 95% ethanol by volume at a mass volume ratio of 1 g:3 ml, reacted for 1 h, filtered, and sterilized at 110° C. for 0.5 h to obtain purified γ-aminobutyric acid.

[0044] Example 3

[0045] A method for extracting purified gamma-aminobutyric acid from ice plant, comprising the following steps:

[0046] (1) placing the ice plant at 35° C. under anaerobic conditions for 2.5 hours, and then placing it at 35° C. under aerobic conditions for 1.5 hours, repeating the above steps three times, crushing and passing through a 60-mesh sieve, adding the sieved ice plant to a 75% by volume ethanol solution at a volume mass ratio of ethanol to ice plant of 9 ml:1 g, and soaking for 35 minutes to obtain an ice plant pre-extract solution;

[0047] (2) extracting the pre-extracted ice plant at 100°C for 8 min each time, extracting at this temperature twice, and concentrating at 55°C for 1.5 h to obtain a crude extract;

[0048] (3) The crude extract was mixed with 92% ethanol by volume at a mass volume ratio of 1 g:2.5 ml, reacted for 1.5 h, filtered, and sterilized at 90° C. for 0.6 h to obtain purified γ-aminobutyric acid.

[0049] Comparative Example 1

[0050] The method in Example 1 was used to extract the purified γ-aminobutyric acid, except that the step (1) of "standing the ice plant at 30° C. under anaerobic conditions for 3 h, then standing it at 30° C. under aerobic conditions for 2 h, and repeating the above steps twice" was omitted.

[0051] Comparative Example 2

[0052] The purified γ-aminobutyric acid was extracted by the method in Example 1, with the only difference being that the step (1) of "place the ice plant at 30°C under anaerobic conditions for 3 h, then place it at 30°C under aerobic conditions for 2 h, and repeat the above steps twice" was replaced by "place the ice plant at -10°C for low temperature treatment for 8 h".

[0053] Comparative Example 3

[0054] The purified γ-aminobutyric acid was extracted by the method in Example 1, except that the step (1) of "adding the sieved ice plant to a 70% ethanol solution with a volume mass ratio of ethanol to ice plant of 8 ml:1 g and soaking for 30 min" was omitted, and the constant temperature extraction time in step (2) was extended to 1 h.

[0055] Comparative Example 4

[0056] The purified γ-aminobutyric acid was extracted by the method in Example 1, except that the content of "extracting the pre-extracted ice plant at a constant temperature of 100° C. for 10 min" in step (2) was replaced by "extracting the pre-extracted ice plant at 50° C. and 150 W ultrasonic wave-assisted for 40 min".

[0057] Test Example 1

[0058] The purified γ-aminobutyric acid was extracted from 1 g of ice plant using the methods of Examples 1-3 and Comparative Examples 1-4.

[0059] Accurately weigh 100 mg of γ-aminobutyric acid standard, dissolve it in distilled water, and dilute to 10 ml to obtain a 10 mg / ml standard solution. Prepare γ-aminobutyric acid standard solutions with concentrations of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 mg / ml. Use distilled water as a blank control, take 1 ml of each, add 50 μL of 2 mol / L AlCl3 solution, mix well, shake at room temperature for 15 min, centrifuge at 12000 r / min for 5 min, take 0.5 ml of the supernatant, add 300 μL of 1 mol / L KOH solution, shake at room temperature 5min, centrifuge at 12000r / min for 5min, take 300μL supernatant, add 500μL, 0.1mol / L, pH10 boric acid buffer solution, 400μL, 6% redistilled phenol solution, mix well, add 600μL, 5% sodium hypochlorite solution, shake well, boil in water bath for 10min, ice bath for 5min, and keep shaking continuously, add 2ml of 60% ethanol solution by volume after blue-green color appears, shake evenly again, measure its absorbance at 645nm wavelength after standing, and draw a standard curve with concentration as horizontal coordinate x and absorbance as vertical coordinate y. The standard curve is as shown in Figure 1 shown.

[0060] Take 1 ml of the purified γ-aminobutyric acid, treat it according to the above method and measure the absorbance at a wavelength of 645 nm. According to the formula: GABA content (mg / g) = C×V / m; C is the value calculated according to the standard curve (mg / ml), V is the volume of the purified γ-aminobutyric acid (ml), and m is the mass of ice plant (g). The test results are shown in Table 1.

[0061] Table 1 GABA determination results

[0062] Group Absorbance Concentration (mg / ml) GABA content (mg / g) Example 1 0.7369 5.2 5.2 Example 2 0.6481 4.9 4.9 Example 3 0.7664 5.3 5.3 Comparative Example 1 0.1453 3.2 3.2 Comparative Example 2 0.4411 4.2 4.2 Comparative Example 3 0.2340 3.5 3.5 Comparative Example 4 0.3523 3.9 3.9

[0063] It can be seen that the content of GABA in the purified γ-aminobutyric acid obtained by extraction of the present invention can reach 4.9 to 5.3 mg / g.

[0064] Test Example 2

[0065] The purified γ-aminobutyric acid obtained in Examples 1-3 and Comparative Examples 1-4 were respectively placed in an environment with a pressure of 0.8 MPa, a feed rate of 0.9 L / min, and a temperature of 120° C. for spray drying to obtain γ-aminobutyric acid products.

[0066] According to the method in "QB / T 4587-2013 γ-aminobutyric acid", the sensory indexes and physicochemical properties of the γ-aminobutyric acid products prepared in Examples 1-3 and Comparative Examples 1-4 were respectively measured, and the contents of arsenic, lead, total bacterial count, coliform group, mold and yeast, and pathogenic bacteria (Salmonella, Shigella and Staphylococcus aureus) in the γ-aminobutyric acid products prepared in Examples 1-3 were measured. The results of the measurements are shown in Tables 2-4.

[0067] Table 2 Sensory indicators of γ-aminobutyric acid products

[0068]

[0069] Table 3 Physical and chemical properties of γ-aminobutyric acid products

[0070]

[0071] Table 4 Hygiene requirements for γ-aminobutyric acid products

[0072]

[0073]

[0074] It can be seen that the γ-aminobutyric acid prepared by the method provided by the present invention has very good quality, and its sensory quality, physical and chemical properties and hygienic conditions all meet the requirements of production and processing, and can be used as a food additive.

[0075] Test Example 3

[0076] 90 SPF C57BL / 6 male mice (purchased from Shanghai Slake Experimental Animal Co., Ltd.) aged 6 weeks and weighing 18-20g were selected and randomly divided into 9 groups (control group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group) after adaptive feeding for 1 week, with 10 mice in each group. Among them, the control group received normal feed, the model group was fed with a high-fat diet (purchased from Nantong Trophy Feed Technology Co., Ltd.), and the remaining groups of mice were fed with γ-aminobutyric acid and high-fat diet prepared in the corresponding examples and comparative examples at a dosage of 2 mg / ml per day. For a period of 5 months.

[0077] After 5 months, the mice were fasted for 12 h, and their body weight, thymus and spleen weight were measured. The organ index of the mice was calculated according to the formula: organ index (%) = organ weight / mouse weight × 100%.

[0078] At the same time, the eyeballs of the mice were removed and blood was collected. The red blood cells were lysed with red blood cell lysis buffer, centrifuged, and resuspended with freshly prepared PBS solution. Cells with cell viability > 98% were selected and the number of cells was 1×106 / ml cell suspension, add the cell suspension into the flow cytometer, mark with Anti-Mouse CD4 FITC, Anti-Mouse CD3e PE-Cy5, Anti-Mouse CD8aPE monoclonal antibody (all purchased from eBioscience, USA) in the dark, sort on the flow cytometer, and sort the CD4 + CD25 + CD127 - / low The regulatory T cells were resuspended in PBS and centrifuged, PBS was removed, 1 ml of 10 μmol / L DCFH-DA was added, and the cells were incubated in a Thermo CO2 incubator at 37°C for 20 min. The cells were washed with PBS three times and detected by flow cytometry. The mitochondrial membrane potential was also detected.

[0079] The mitochondrial membrane potential was measured using a test kit (purchased from Biotech Research Institute). The results are shown in Table 5 below.

[0080] Table 5 Determination results of mouse immune indicators

[0081]

[0082] As shown in Table 5, the results of the model group showed that the immune function of mice fed with a high-fat diet was damaged to a certain extent; compared with the model group, the degree of damage to the body of mice fed with the γ-aminobutyric acid extracted in Examples 1-3 of the present invention was significantly reduced, and the thymus index and spleen index of the mice were close to the levels of normal mice. That is, the γ-aminobutyric acid extracted in the present invention has a good effect of improving immune function and enhancing immunity.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting purified gamma-aminobutyric acid from ice plant, characterized in that: The steps include: (1) treating the ice plant in an anaerobic-aerobic alternating manner, crushing, and soaking to obtain an ice plant pre-extract solution; (2) extracting the pre-extracted ice plant at a constant temperature, and concentrating the extract to obtain a crude extract; (3) removing impurities from the crude extract, sterilizing it, and obtaining a purified γ-aminobutyric acid product; The temperature of the anaerobic-aerobic alternating treatment in step (1) is 30-40° C., the number of the anaerobic-aerobic alternating treatment is 2-4 times, and the method of the anaerobic-aerobic alternating treatment is: placing the ice plant under anaerobic conditions for 2-3 hours, and then placing it under aerobic conditions for 1-2 hours; The immersion in step (1) is carried out using ethanol at a temperature of 25 to 30° C. and a immersion time of 30 to 40 minutes; The temperature of the constant temperature extraction in step (2) is 90-100° C., the time of the constant temperature extraction is 5-10 min / time, and the number of constant temperature extractions is 2-4 times.

2. The method according to claim 1, characterized in that: After the pulverization in step (1), the product is sieved through a 50-60 mesh screen.

3. The method according to claim 1, characterized in that: The volume fraction of the ethanol is 70-80%, and the volume mass ratio of the ethanol to the ice plant is 8-10 ml:1 g.

4. The method according to claim 1, characterized in that The concentration temperature in step (2) is 50-60° C., and the concentration time is 1-2 hours.

5. The method according to claim 1, characterized in that: The method for removing impurities in step (3) is to mix the crude extract with ethanol, react for 1 to 2 hours, filter, and obtain a de-impurified product.

6. The method according to claim 5, characterized in that The volume fraction of the ethanol is 90-95%, and the mass volume ratio of the crude extract to the ethanol is 1g:2-3ml.

7. The method according to claim 1, characterized in that The sterilization temperature in step (3) is 90-110° C., and the sterilization time is 0.5-1 h.

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