Preparation method of perigord truffle extract as well as product and application of perigord truffle extract

Abstract

The invention relates to a preparation method of a perigord truffle extract as well as a product and application of the perigord truffle extract. The preparation method comprises the following steps: mixing perigord truffle with an enzyme solution for enzymolysis to obtain enzymatic hydrolysate; and mixing the enzymatic hydrolysate with an extracting agent, treating in a pulsed electric field, extracting, centrifuging, taking supernate, and filtering to obtain the traditional Chinese medicine composition. The perigord truffle extract is prepared through a specific extraction method, and the prepared perigord truffle extract has good antioxidant and anti-inflammatory effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of substance extraction, and relates to a preparation method, product and application of a black truffle extract. Background Art

[0002] Black truffle is a rare and expensive fungus. Its unique and strong flavor, as well as rich nutritional components and bioactive substances, show great application potential in the fields of food, medicine and cosmetics. The antioxidant and moisturizing properties of black truffle extract make it an excellent choice for cosmetic raw materials. Skin care products added with black truffle extract can help the skin resist free radical damage, delay aging, improve the water content and gloss of the skin, and have broad development prospects in the high-end cosmetic market.

[0003] At present, organic solvents such as ethanol and acetone are commonly used to soak and extract black truffles. However, this method has low extraction efficiency and is difficult to achieve efficient extraction of some active ingredients with large polarity differences, resulting in low content of target components in the extract and unstable product quality. The ultrasonic-assisted extraction method can accelerate the dissolution of components in black truffles through the cavitation effect of ultrasonic waves and improve the extraction efficiency. However, if the intensity and time of ultrasonic waves are not properly controlled, it may damage the structures of some active ingredients in black truffles and affect the biological activity of the extract. Moreover, when this method is used alone, the extraction selectivity for complex components is limited and there are many impurities in the extract.

[0004] In view of the deficiencies in the existing preparation methods of black truffle extract, it is of great practical significance to develop a preparation method that is efficient, safe, environmentally friendly and can retain the active ingredients of black truffles to the greatest extent. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a preparation method, product and application of a black truffle extract.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted:

[0007] In the first aspect, the present invention provides a preparation method of a black truffle extract, and the preparation method includes:

[0008] (1) Mix black truffles with an enzyme solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0009] (2) Mix the enzymatic hydrolysate with an extractant, treat it under pulsed electric field, extract, centrifuge to obtain the supernatant, and filter to obtain the product.

[0010] The black truffle is pretreated with enzymes to obtain a black truffle enzymatic hydrolysate rich in highly active substances. Using the high-voltage pulsed electric field extraction method, through pulsed electric field cell wall breaking, the cell membrane and cell wall potentials of the treated cells can be instantly disrupted, changing their permeability. It can even penetrate the cell membrane and cell wall to cause irreversible damage, resulting in the outflow of cell contents and greatly improving the extraction rate of effective substances. The extraction process is low-temperature, fast, green, and has no organic residues.

[0011] Preferably, the material-liquid ratio of the black truffle to the enzyme solution is 1 g:(5 - 20) mL.

[0012] Specific point values in (5 - 20) can be selected as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0013] Preferably, the temperature of the enzymatic hydrolysis is 25 - 35 °C and the time is 20 - 40 min.

[0014] The temperature can be selected as 25 °C, 28 °C, 30 °C, 32 °C, 35 °C, etc., and the time can be selected as 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0015] Preferably, the enzyme includes cellulase, papain, and chitinase.

[0016] Cellulase, papain, and chitinase have a certain cooperation in the above effects and can promote the extraction of functional substances.

[0017] Preferably, the addition amount of the enzyme is 5 - 15 U / mL, such as 5 U / mL, 6 U / mL, 7 U / mL, 8 U / mL, 9 U / mL, 10 U / mL, 11 U / mL, 12 U / mL, 13 U / mL, 14 U / mL, 15 U / mL, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0018] Preferably, the enzyme activity ratio of cellulase, papain, and chitinase is (3 - 6):(1 - 4):(1 - 5).

[0019] The specific point values in (3-6) can all be selected from 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, etc. The specific point values in (1-4) can all be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc. The specific point values in (1-5) can all be selected from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0020] Preferably, the extractant includes glycerol, 1,2-pentanediol, gallic acid, citric acid and water.

[0021] Glycerol, 1,2-pentanediol, gallic acid and citric acid can promote the extraction of active substances, and when several components are added simultaneously, the anti-inflammatory and moisturizing effects of the obtained extract are optimal.

[0022] Preferably, the extractant includes, by mass, 60-80 parts of glycerol, 1-5 parts of 1,2-pentanediol, 2-10 parts of gallic acid, 1-15 parts of citric acid and 6-10 parts of water.

[0023] The mass parts of glycerol can be selected from 60 parts, 62 parts, 65 parts, 68 parts, 70 parts, 72 parts, 74 parts, 76 parts, 78 parts, 80 parts, etc. The mass parts of 1,2-pentanediol can be selected from 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, etc. The mass parts of gallic acid can be selected from 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc. The mass parts of citric acid can be selected from 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, etc. The mass parts of water can be selected from 6 parts, 6.5 parts, 7 parts, 7.5 parts, 8 parts, 8.5 parts, 9 parts, 9.5 parts, 10 parts, etc. Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0024] Preferably, the volume ratio of the enzymatic hydrolysate to the extractant is 1:(10-50).

[0025] The specific point values in (10-50) can all be selected from 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, etc. Other specific point values within the above numerical ranges can be selected, and will not be elaborated one by one here.

[0026] Preferably, the field strength of the pulsed electric field is 30-50 kv / m, and the number of electric shocks is 80-160 times.

[0027] The electric field strength can be selected as 30 kv / m, 32 kv / m, 35 kv / m, 38 kv / m, 40 kv / m, 42 kv / m, 45 kv / m, 48 kv / m, 50 kv / m, etc., and the number of times can be selected as 80 times, 85 times, 90 times, 95 times, 100 times, 105 times, 110 times, 115 times, 120 times, 125 times, 130 times, 135 times, 140 times, 145 times, 150 times, 155 times, 160 times, etc. Other specific point values within the above numerical range can be selected, and will not be elaborated one by one here.

[0028] Preferably, the temperature for extraction is 25 - 30 °C, and the extraction time is 50 - 70 min.

[0029] The temperature can be selected as 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, etc., and the time can be selected as 50 min, 52 °C, 55 °C, 57 °C, 58 °C, 60 °C, 62 °C, 65 °C, 68 °C, 70 °C, etc. Other specific point values within the above numerical range can be selected, and will not be elaborated one by one here.

[0030] Preferably, the extraction is carried out under stirring conditions, and the rotation speed of the stirring is 400 - 600 rpm.

[0031] The rotation speed can be selected as 400 rpm, 420 rpm, 450 rpm, 480 rpm, 500 rpm, 520 rpm, 550 rpm, 580 rpm, 600 rpm, etc. Other specific point values within the above numerical range can be selected, and will not be elaborated one by one here.

[0032] Preferably, the rotation speed of the centrifugation is 12000 - 15000 rpm, and the time is 10 - 20 min.

[0033] The rotation speed can be selected as 12000 rpm, 12500 rpm, 13000 rpm, 13500 rpm, 14000 rpm, 14500 rpm, 15000 rpm, etc., and the time can be selected as 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc. Other specific point values within the above numerical range can be selected, and will not be elaborated one by one here.

[0034] In a second aspect, the present invention provides a black truffle extract prepared by the preparation method of the black truffle extract according to the first aspect.

[0035] In a third aspect, the present invention provides an application of the black truffle extract according to the second aspect in the preparation of a product with moisturizing or anti - inflammatory efficacy.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention utilizes the high - voltage pulsed - electric - field extraction method. Through pulsed - electric - field cell wall breaking, it can instantaneously disrupt the cell - membrane and cell - wall potentials of the cells to be processed, change their permeability. It can even reach the level of breaking through the cell membrane and cell wall to cause irreversible damage, resulting in the outflow of cell contents, greatly improving the extraction rate of effective substances. The extraction process is low - temperature, fast, green, and has no organic residues.

[0038] 2. The present invention combines the high - voltage pulsed - electric - field extraction technology with the eutectic extraction method to extract black truffle. By studying the black - truffle extraction technology and conducting efficacy verification of black - truffle extracts, it develops black - truffle - extract skin - care products with good antioxidant and anti - inflammatory effects, which can not only effectively combat skin oxidation caused by internal and external factors of the body but also maintain skin health. Specific Embodiments

[0039] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in combination with the preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0040] The sources of the active ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the active ingredients are shown, and the necessary auxiliary ingredients contained in other commercially available raw materials are not elaborated):

[0041] Cellulase is a product purchased from Shanghai Yuanye Company with the product name S10041;

[0042] Papain is a product purchased from Shanghai Yuanye Company with the product name S27241;

[0043] Chitinase is a product purchased from Shanghai Yuanye Company with the product name S10178.

[0044] Preparation Example 1

[0045] This preparation example provides an extractant. The extractant includes 70 parts by mass of glycerol, 2.5 parts of 1,2 - pentanediol, 6 parts of gallic acid, 12 parts of citric acid, and 9 parts of water.

[0046] Its preparation method is: physically mix the preparation raw materials to obtain it.

[0047] Preparation Example 2

[0048] This preparation example provides an extractant. The extractant includes 60 parts by mass of glycerol, 5 parts of 1,2 - pentanediol, 10 parts of gallic acid, 15 parts of citric acid, and 10 parts of water.

[0049] The preparation method refers to Preparation Example 1.

[0050] Preparation Example 3

[0051] This preparation example provides an extractant, which includes, by mass fraction, 80 parts of glycerol, 2 parts of 1,2-pentanediol, 2 parts of gallic acid, 4 parts of citric acid, and 6 parts of water.

[0052] The preparation method refers to Preparation Example 1.

[0053] Preparation Example 4

[0054] This preparation example provides an extractant, which includes, by mass fraction, 5 parts of 1,2-pentanediol, 10 parts of gallic acid, 15 parts of citric acid, and 3 parts of water.

[0055] The preparation method refers to Preparation Example 1.

[0056] Preparation Example 5

[0057] This preparation example provides an extractant, which is different from Preparation Example 1 only in that it does not contain 1,2-pentanediol, and the reduced mass is proportionally distributed to the masses of glycerol, gallic acid, and citric acid, while other components and contents remain unchanged.

[0058] The preparation method refers to Preparation Example 1.

[0059] Preparation Example 6

[0060] This preparation example provides an extractant, which is different from Preparation Example 1 only in that it does not contain gallic acid, and the reduced mass is proportionally distributed to the masses of glycerol, 1,2-pentanediol, and citric acid, while other components and contents remain unchanged.

[0061] The preparation method refers to Preparation Example 1.

[0062] Preparation Example 7

[0063] This preparation example provides an extractant, which is different from Preparation Example 1 only in that it does not contain citric acid, and the reduced mass is proportionally distributed to the masses of glycerol, 1,2-pentanediol, and gallic acid, while other components and contents remain unchanged.

[0064] The preparation method refers to Preparation Example 1.

[0065] Preparation Example 8

[0066] This preparation example provides an extractant, which includes, by mass fraction, 4 parts of betaine, 1 part of propylene glycol, and 15 parts of glycerol.

[0067] The preparation method refers to Preparation Example 1.

[0068] Example 1

[0069] This example provides a preparation method of a black truffle extract, and the preparation method includes:

[0070] (1) Wash the black truffles, dry them at 60 °C, and pulverize them with a grinder to obtain black truffle dry powder;

[0071] (2) Mix the black truffle dry powder with the enzyme solution at a solid-liquid ratio of 1 g:12 mL, and treat it at 30 °C for 30 min to obtain an enzymolysis solution. The enzyme addition amount is 10 U / mL, and the enzyme activity ratio of cellulase, papain, and chitinase is 4:2:4;

[0072] (3) Mix the enzymolysis solution with the extractant described in Preparation Example 1 at a volume ratio of 1:30, and treat it under pulsed electric field. The field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under stirring conditions of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain the product.

[0073] Example 2

[0074] This example provides a method for preparing a black truffle extract, and the preparation method includes:

[0075] (1) Wash the black truffles, dry them at 60 °C, and pulverize them with a grinder to obtain black truffle dry powder;

[0076] (2) Mix the black truffle dry powder with the enzyme solution at a solid-liquid ratio of 1 g:5 mL, and treat it at 35 °C for 20 min to obtain an enzymolysis solution. The total enzyme addition amount is 5 U / mL, and the enzyme activity ratio of cellulase, papain, and chitinase is 5:1:2;

[0077] (3) Mix the enzymolysis solution with the extractant described in Preparation Example 2 at a volume ratio of 1:50, and treat it under pulsed electric field. The field strength of the pulsed electric field is 30 kv / m, the number of electric shocks is 160 times, extract at 25 °C under stirring conditions of 400 rpm for 70 min, centrifuge at 12000 rpm for 20 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain the product.

[0078] Example 3

[0079] This example provides a method for preparing a black truffle extract, and the preparation method includes:

[0080] (1) Wash the black truffles, dry them at 60 °C, and pulverize them with a grinder to obtain black truffle dry powder;

[0081] (2) Mix the black truffle dry powder with the enzyme solution at a solid-liquid ratio of 1 g:20 mL, and treat it at 25 °C for 40 min to obtain an enzymolysis solution. The total enzyme addition amount is 15 U / mL, and the enzyme activity ratio of cellulase, papain, and chitinase is 3:4:5;

[0082] (3) Mix the enzymatic hydrolysate with the extractant described in Preparation Example 3 at a volume ratio of 1:10, and treat it under pulsed electric field. The field strength of the pulsed electric field is 50 kv / m, the number of electric shocks is 80 times, extract at 30 °C under stirring conditions of 600 rpm for 50 min, centrifuge at 15000 rpm for 10 min, take the supernatant, and filter through a 0.22 μm membrane to obtain the product.

[0083] Example 4

[0084] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (2) is "mix the black truffle dry powder with the enzyme solution at a material-liquid ratio of 1 g:12 mL, treat at 30 °C for 30 min to obtain the enzymatic hydrolysate, the total amount of enzyme added is 10 U / mL, and the enzyme activity ratio of cellulase to papain is 2:1".

[0085] Keep other operations unchanged.

[0086] Example 5

[0087] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (2) is "mix the black truffle dry powder with the enzyme solution at a material-liquid ratio of 1 g:12 mL, treat at 30 °C for 30 min to obtain the enzymatic hydrolysate, the total amount of enzyme added is 10 U / mL, and the enzyme activity ratio of cellulase to chitinase is 1:1".

[0088] Keep other operations unchanged.

[0089] Example 6

[0090] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (2) is "mix the black truffle dry powder with the enzyme solution at a material-liquid ratio of 1 g:12 mL, treat at 30 °C for 30 min to obtain the enzymatic hydrolysate, the total amount of enzyme added is 10 U / mL, and the enzyme activity ratio of papain to chitinase is 1:2".

[0091] Keep other operations unchanged.

[0092] Example 7

[0093] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (3) is "mix the enzymatic hydrolysate with the extractant described in Preparation Example 4 at a volume ratio of 1:30, and treat it under pulsed electric field. The field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under stirring conditions of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter through a 0.22 μm membrane to obtain the product".

[0094] Keep other operations unchanged.

[0095] Example 8

[0096] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (3) is "mix the enzymolysis solution with the extractant described in Preparation Example 5 at a volume ratio of 1:30, treat it under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain it."

[0097] Other operations remain unchanged.

[0098] Example 9

[0099] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (3) is "mix the enzymolysis solution with the extractant described in Preparation Example 6 at a volume ratio of 1:30, treat it under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain it."

[0100] Other operations remain unchanged.

[0101] Example 10

[0102] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (3) is "mix the enzymolysis solution with the extractant described in Preparation Example 7 at a volume ratio of 1:30, treat it under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain it."

[0103] Other operations remain unchanged.

[0104] Example 11

[0105] This example provides a method for preparing a black truffle extract, which is only different from Example 1 in that step (3) is "mix the enzymolysis solution with the extractant described in Preparation Example 8 at a volume ratio of 1:30, treat it under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter it through a 0.22 μm membrane to obtain it."

[0106] Other operations remain unchanged.

[0107] Comparative Example 1

[0108] This comparative example provides a method for preparing a black truffle extract, and the preparation method includes:

[0109] (1) Wash the black truffles, dry them at 60 °C, and after pulverizing with a grinder, obtain black truffle dry powder;

[0110] (2) Mix the black truffle dry powder and water at a solid-liquid ratio of 1 g:12 mL, and treat at 30 °C for 30 min;

[0111] (3) Mix the product obtained in step (2) and the extractant described in Preparation Example 1 at a volume ratio of 1:30, treat under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter through a 0.22 μm membrane to obtain.

[0112] Comparative Example 2

[0113] This comparative example provides a method for preparing a black truffle extract, and the difference from Example 1 is only that step (3) is "mix the enzymolysis solution and the extractant described in Preparation Example 1 at a volume ratio of 1:30, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter through a 0.22 μm membrane".

[0114] Other operations remain unchanged.

[0115] Comparative Example 3

[0116] This comparative example provides a method for preparing a black truffle extract, and the difference from Example 1 is only that step (3) is "mix the enzymolysis solution and water at a volume ratio of 1:30, treat under pulsed electric field, the field strength of the pulsed electric field is 40 kv / m, the number of electric shocks is 120 times, extract at 28 °C under the stirring condition of 500 rpm for 60 min, centrifuge at 13000 rpm for 15 min, take the supernatant, and filter through a 0.22 μm membrane to obtain.

[0117] Other operations remain unchanged.

[0118] Test Example 1

[0119] Protein content test

[0120] The test method refers to the total protein quantification kit (BCA method), and the kit is purchased from Nanjing Jiancheng Bioengineering Institute, with the product number A045-4-2.

[0121] The test results are shown in Table 1.

[0122] Table 1

[0123] Protein content (%) Example 1 25.34 Example 2 23.16 Example 3 22.47

[0124] As can be seen from the data in Table 1, the black truffle extract prepared by the present invention has a high protein content.

[0125] Test Example 2

[0126] Moisturizing effect test

[0127] Quantitative real-time PCR (qPCR) was used to measure the gene expression level of aquaporin 3 (AQP3). The specific test method was as follows:

[0128] After the human skin fibroblast BJ cell line in the 6-well plate grew to about 70% confluence, the medium was replaced with a medium containing 10% fetal bovine serum (FBS). After starvation culture for 24 hours, the medium was replaced with a medium containing the test samples at different concentrations and treated for 24 hours. TRIzol reagent was used to extract RNA from the cells. After measuring the RNA concentration, the mRNA was reverse transcribed into cDNA, which was used as a template for fluorescence quantitative PCR to detect the gene expression of AQP3 synthesis in the test molecules at the gene level.

[0129] Each sample was tested independently 3 times, and the results were presented as mean ± standard deviation. The negative control was the cells treated with the medium containing 10% FBS for 24 hours; the reference calibration gene was the internal reference gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene).

[0130] Table 2

[0131]

[0132] As can be seen from the data in Table 2, the black truffle extract prepared by the present invention has a better moisturizing effect. The choice of enzyme has a significant impact on the moisturizing effect of the extract, and the treatments with cellulase, papain and chitinase have a certain synergistic effect in enhancing the moisturizing effect of the extract. Moreover, the choice of the components of the extractant also affects the moisturizing effect of the product.

[0133] Test Example 3

[0134] Anti-inflammatory effect test

[0135] Quantitative real-time PCR (qPCR) was used to measure the gene expression levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). The specific test method was as follows:

[0136] After the human skin fibroblast BJ cell line in the 6-well plate grew to about 70% confluence, the medium was replaced with a medium containing 10% FBS. After starvation culture for 24 hours, the medium was replaced with a medium containing 1% test sample and 2 μg / mL lipopolysaccharide (LPS) and treated for 24 hours. TRIzol reagent was used to extract RNA from the cells. After measuring the RNA concentration, the mRNA was reverse transcribed into cDNA, which was used as a template for fluorescence quantitative PCR to detect the synthetic expression of the molecule to be tested at the gene level. Each sample was tested independently 3 times, and the results were presented in the form of mean ± standard deviation. The cells in the model group were treated with a medium containing 10% FBS with LPS for 24 hours; the cells in the positive control group were treated with a medium containing 0.2 mg / mL dexamethasone for 24 hours; the cells in the negative control group were treated with a medium containing 10% FBS for 24 hours; the reference calibration gene was the internal reference gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene).

[0137] Table 3

[0138]

[0139] As can be seen from the data in Table 3, the truffle extract prepared by the present invention has excellent anti-inflammatory effects. The selection of enzymes has a significant impact on the anti-inflammatory effects of the extract, and the treatments with cellulase, papain, and chitinase have certain synergistic effects in enhancing the anti-inflammatory effects of the extract. Moreover, the selection of the components of the extractant also affects the anti-inflammatory effects of the product.

[0140] The applicant declares that the present invention uses the above embodiments to illustrate a preparation method, its products, and applications of a truffle extract of the present invention. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0141] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0142] In addition, it should be noted that in the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for preparing a black truffle extract, characterized in that, The preparation method includes: (1) Mixing and enzymolyzing black truffle with an enzyme solution to obtain an enzymolyzed solution; (2) Mixing the enzymolyzed solution with an extractant, treating it under pulsed electric field, extracting, centrifuging to obtain the supernatant, and filtering to obtain the product.

2. The preparation method of the black truffle extract according to claim 1, characterized in that, The material-liquid ratio of the black truffle to the enzyme solution is 1 g:(5 - 20) mL; Preferably, the temperature of the enzymolysis is 25 - 35 °C and the time is 20 - 40 min.

3. The preparation method of the black truffle extract according to claim 1 or 2, characterized in that, The enzyme includes cellulase, papain and chitinase; Preferably, the addition amount of the enzyme is 5 - 15 U / mL; Preferably, the enzyme activity ratio of the cellulase, papain and chitinase is (3 - 6):(1 - 4):(1 - 5).

4. The preparation method of the black truffle extract according to any one of claims 1-3, characterized in that, The extractant includes glycerol, 1,2-pentanediol, gallic acid, citric acid and water; Preferably, the extractant includes 60 - 80 parts by mass of glycerol, 1 - 5 parts by mass of 1,2-pentanediol, 2 - 10 parts by mass of gallic acid, 1 - 15 parts by mass of citric acid and 6 - 10 parts by mass of water.

5. The preparation method of the black truffle extract according to any one of claims 1-4, characterized in that, The volume ratio of the enzymolyzed solution to the extractant is 1:(10 - 50).

6. The preparation method of the black truffle extract according to any one of claims 1-5, characterized in that, The field strength of the pulsed electric field is 30 - 50 kv / m and the number of electric shock times is 80 - 160 times.

7. The preparation method of the black truffle extract according to any one of claims 1-6, characterized in that, The temperature of the extraction is 25 - 30 °C and the time of the extraction is 50 - 70 min.

8. The preparation method of the black truffle extract according to any one of claims 1-7, characterized in that, The extraction is carried out under stirring conditions, and the rotation speed of the stirring is 400 - 600 rpm.

9. A black truffle extract prepared by the preparation method of the black truffle extract according to any one of claims 1 - 8.

10. An application of the black truffle extract according to claim 9 in the preparation of a product with moisturizing or anti-inflammatory effects.

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