Preparation method and application of red date paste with neuroprotective peptide fragment

Abstract

The invention provides a preparation method and application of red date paste with a neuroprotective peptide fragment. An innovative process capable of retaining bioactive substances of the red dates to the greatest extent is developed by deeply researching various pretreatment technologies such as air flash evaporation, ultrasonic light cooking and high-pressure cooking, the content of adenosine cyclophosphate in the red date paste prepared by the process is up to 370 + / -14 micrograms per gram, the protein content is up to 1.12%, and the red date paste is suitable for large-scale production. And a key peptide fragment with a neuroprotective effect is found in the red date polypeptide. In a PC12 cell model induced by hydrogen peroxide, Cyclo-GP and Ala-Pro-Ile show a remarkable cell protection effect, and the Cyclo-GP and Ala-Pro-Ile are two of the five candidate peptide fragments with the most remarkable neuroprotection function effects. The preparation process of the red date paste is simple and convenient to operate, high in extraction efficiency and low in cost, not only provides a new technical direction for deep processing of red dates, but also lays a solid foundation for developing red date products with a nerve protection function.

Description

Technical Field

[0001] The present invention relates to the technical field of red date processing. More specifically, the present invention relates to the field of health foods. Five peptide segments are found in red date paste, which involves a production process of natural red date paste, and also involves a pretreatment, processing, detection and industrial production method of red date paste with peptide segments having neuroprotective functions.

Technical Background

[0002] Red dates have extremely rich nutritional value, containing proteins, fats, amino acids, carbohydrates, vitamins, organic acids, etc. required by the human body, and have important research value. Steaming and drying red dates three times is a traditional production method, but it takes a long time and the processing technology is cumbersome. On the basis of the classic red date preparation process, modern process improvements such as air flash evaporation, ultrasonic light cooking, and high-pressure steaming and boiling not only save time costs, but also maximize the retention of the nutritional components of red dates. After detecting the components of the red date paste produced by this process, various nutritional and medicinal components in red dates can be better released through this production method, thereby enhancing the medicinal effects and nutritional value of red dates. Amino acid composition is one of the important components of the nutritional and functional characteristics of red dates, and it is of practical significance to study the amino acid composition of the main cultivated red dates in Xinjiang. Cyclic adenosine monophosphate (cAMP) in red dates has been measured to have the highest content among higher plants. It belongs to derivatives of nucleotides. As a common second messenger in cells, it jointly participates in the regulation of intracellular physiological and biochemical processes, and has functions such as lowering blood sugar, anti-allergy, inhibiting cardiovascular diseases, and inhibiting the growth of cancer cells. It is an important bioactive substance in red date fruits.

[0003] There are many varieties of red dates. Among them, medicinal amino acids, branched-chain amino acids, aromatic amino acids, and umami amino acids in red dates are relatively rich. Nine amino acids, namely aspartic acid, glutamic acid, glycine, methionine, leucine, tyrosine, phenylalanine, lysine, and arginine, are listed as pharmacodynamic amino acids because they have some special medical and health care effects in humans and animals.

[0004] The traditional red date paste preparation process destroys the content of its own bioactive substances, causing serious waste of red date resources and being difficult to meet the requirements of deep processing products.

Summary of the Invention

[0005] The purpose of the present invention is to solve the existing deficiencies and provide a preparation method and application of red date paste with neuroprotective peptide segments, which is a red date paste with efficient nutrition, simple preparation, time-saving and fast speed, having neuroprotective functions and healthy and green health care effects, including a comparative example and an example. The existing red date paste preparation method is marked as the comparative example, and the red date paste prepared by the present invention is the example, which specifically includes the following steps;

[0006] Step 1: Raw material preparation: Select red dates that are round on the surface, bright in color, fully ripe, and produced in Xinjiang (with a moisture content of 25%-45%, being Xinjiang Jun dates or Xinjiang Gray dates), and take them out from the 4°C refrigeration device;

[0007] Step 2: Soaking and cleaning

[0008] Take the materials, with a material ratio of one part by mass of red dates to two parts by mass of water, soak them, aiming to clean the soil and impurities on the surface;

[0009] Step 3: Air drying

[0010] Lay the cleaned red dates flat in a ventilated place to let the moisture evaporate completely;

[0011] Step 4: Air flash evaporation

[0012] For the red dates without moisture on the surface, the mass ratio of red dates to water is 1:2;

[0013] Then place them in a steam device with a pot temperature of 95-110°C for air flash evaporation treatment for 1-3 minutes. When a large amount of steam emerges from the steamer, that is, when the pot temperature reaches 100°C, place the dates and water on the steamer and flash evaporate for 1-3 minutes;

[0014] Step 5: Ultrasonic light ripening

[0015] Place them in an ultrasonic cleaner for ultrasonic treatment, with the time set to 10-40 minutes, the temperature to 20-40°C, and the power to 60% (rated power 480V);

[0016] Step 5: High-pressure steaming

[0017] In a high-pressure pot, set the pressure to 0.12-0.25 MPa for high-pressure steaming for 10-15 minutes. Further, set the pressure to 0.15 MPa, and start timing for 10-15 minutes when the temperature reaches 125°C;

[0018] Wait until the pressure in the pot drops to normal pressure, take out the dates, remove the date skins and pits, and use a homogenizer to break and stir them together with the date water to obtain date paste with a certain water content.

[0019] Preferably, the soaking time of red dates and water with a mass ratio of 1:2 is 30 minutes,

[0020] Preferably, before high-pressure steaming the dates, pre-treat the dates, and perform cell disruption treatment on the pitted dates by ultrasonic treatment, which is beneficial to the release of intracellular active substances. The ultrasonic light ripening treatment is 40 minutes,

[0021] Preferably, the jujubes are subjected to a high-pressure treatment stage. Based on a pressure setting of 0.15 MPa, when the temperature in the pressure cooker rises to 125 °C, start timing for 15 minutes. The prepared jujube paste has a strong aroma.

[0022] Step 6: Prepare a jujube paste sample by the conventional process and compare it with the jujube paste treated by air flash evaporation, ultrasonic light cooking, and high-pressure steaming.

[0023] The application of red jujube paste with neuroprotective peptide segments in the preparation of neuroprotective functional red jujube paste granule products and in the preparation of neuroprotective functional red jujube paste beverage products.

[0024] The beneficial effects of the present invention are as follows: During the processes of air flash evaporation, ultrasonic light cooking, and high-pressure steaming of red jujubes, a series of chemical reactions such as dehydration, degradation, and oxidation occur between the amino acids and reducing sugar hydroxyl compounds rich in the red jujubes themselves at a certain temperature and time, forming a unique aroma. While the jujube paste forms a jujube aroma, the macromolecular substances contained in the jujubes are degraded into small molecules. The red jujube paste prepared by the present invention is rich in various nutrients required by the human body. Five peptide segments, namely Ala-Pro-Ile, Ile-Pro-Phe, cyclo(Gly-Pro), cyclo(Phe-Ala), and PyroGlu-Pro, are obtained through separation and purification and other means. It is found that these five peptide segments are brand new and have a role in neuroprotection in in vitro experiments. Experiments show that the content of cyclic adenosine monophosphate (cAMP) in the red jujube paste prepared by this process is as high as 370 ± 14 μg / g, the protein content in the red jujube paste prepared by this process is as high as 1.12%, and key peptide segments with neuroprotective effects are found in the red jujube polypeptides, such as Ala-Pro-Ile, Ile-Pro-Phe, cyclo(Gly-Pro), cyclo(Phe-Ala), and PyroGlu-Pro, etc. In the PC12 cell model induced by hydrogen peroxide, Cyclo-GP and Ala-Pro-Ile show significant cell protection effects, and the cell survival rates reach 88.83% and 78.73% respectively, which are the two with the most significant neuroprotective functional effects among the five candidate peptide segments.

[0025] The red jujubes involved in the present invention can not only be used for first-class red jujubes, but also be suitable for the processing and preparation of substandard jujubes, effectively reducing the loss of substandard red jujubes, improving the added value of products, and enhancing the competitiveness of similar products in the market. In addition, the equipment used during the processing of the present invention has a low cost, is green and environmentally friendly during the processing and production process, has low energy consumption, occupies a small space area, does not add any chemical agents and food additives during the whole process of preparing jujube paste, and is natural and healthy.

[0026] The red date paste preparation process of the present invention is simple to operate, has high extraction efficiency and low cost. It not only provides a new technical direction for the deep processing of red dates, but also lays a solid foundation for the development of red date products with neuroprotective functions (such as red date peptide granules and other beverages). In addition, by comprehensively analyzing the change rules of the content of bioactive substances during the deep processing of red dates, the present invention provides important technical references for the subsequent application of red date paste, and is expected to promote the wide application of red date products in the field of health foods.

Description of the Drawings

[0027] Figure 1 For the amino acid content rates of date pastes in different embodiments;

[0028] Figure 2 For the contents of bioactive substances in different embodiments;

[0029] Figure 3 For the sensory evaluation results of different embodiments of date paste;

[0030] Figure 4 For the PCA diagrams of electronic noses of different date paste embodiments;

[0031] Figure 5 For the loading diagrams of different date paste embodiments;

[0032] Figure 6 For the chromatogram of Example 6;

[0033] Figure 7 For the volatile aroma components of GC-MS in Example 6;

[0034] Figure 8 For the DPPH scavenging rate of date paste;

[0035] Figure 9 For the DPPH scavenging rates of VC standard and Example 4;

[0036] Figure 10 For the ABTS+ scavenging rate of date paste;

[0037] Figure 11 For the ABTS + scavenging rate;

[0038] Figure 12 For the ·OH free radical scavenging rate of date paste;

[0039] Figure 13 For the ·OH free radical scavenging rates of VC standard and Example 4;

[0040] Figure 14 For the O2 - free radical scavenging rate;

[0041] Figure 15 For the VC standard product and O2 of Example 4 - Free radical scavenging rate;

[0042] Figure 16 Effect of red date puree concentration on the viability toxicity of PC12 cells. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the model group, #P<0.05;

[0043] Figure 17 Effect of red date puree concentration on the survival rate of H2O2-induced oxidative damage of PC12 cells. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the model group, #P<0.05;

[0044] Figure 18 Protection of five peptide segments against hydrogen peroxide-induced PC12 cells. The concentration of peptide segments is 200 μg / ml. Among them, compared with the control group (control), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Compared with the model group (model), #P<0.05, ##P<0.01, P<0.001, #P<0.0001;

[0045] Figure 19 Process flow chart for preparing red date puree particles;

[0046] Figure 20 Red date particles;

[0047] Figure 21 Process flow chart for preparing red date donkey-hide gelatin peptide beverage;

[0048] Figure 22 Red date donkey-hide gelatin peptide beverage.

Specific implementation manners

[0049] The following is to make the purpose, processing parameters and advantages of the present invention clearer. The present invention will be further described and explained below in combination with embodiments.

[0050] Example 1 (comparative example):

[0051] (1) Raw material preparation: Select red dates with round surface, bright color and origin in Xinjiang, and take them out from the 4°C refrigeration device;

[0052] (2) Soaking and cleaning

[0053] Take the materials, with a material ratio of one part by mass of red dates to two parts by mass of water, soak them, aiming to clean the soil and impurities on the surface;

[0054] (3) Air drying

[0055] The washed red dates are laid flat in a ventilated place to evaporate the moisture completely;

[0056] (4) Preparation of date paste without removing the pits

[0057] Take the red dates with no moisture on the surface, without removing the pits, and the mass ratio of red dates to water is 1:2;

[0058] (5) High-pressure treatment for 15 minutes

[0059] In the autoclave, set the pressure to 0.15 MPa. Start timing for 15 minutes when the temperature reaches 125 °C, and then cool it to room temperature;

[0060] (6) Preparation of date paste

[0061] Wait until the pressure in the autoclave drops to atmospheric pressure, take out the dates, remove the skins and pits, and use a homogenizer to break and stir them together with the date water to obtain date paste with a certain water content.

[0062] (7) Preservation

[0063] Place the prepared date paste sample in a sterile bag and store it frozen in a -20 °C refrigerator.

[0064] Example 2 (control example):

[0065] (1) Raw material preparation: Select red dates with round surfaces, bright colors, and the place of origin being Xinjiang. Take them out from the 4 °C refrigeration device;

[0066] (2) Soaking and cleaning

[0067] Take the materials, with a material ratio of one part of red dates to two parts of water, soak them to clean the soil and impurities on the surface;

[0068] (3) Drying

[0069] The washed red dates are laid flat in a ventilated place to evaporate the moisture completely;

[0070] (4) Preparation of date paste with pits removed

[0071] Take the red dates with no moisture on the surface, remove the pits, cut them into pieces, and the mass ratio of red dates to water is 1:2;

[0072] (5) High-pressure treatment

[0073] In the autoclave, set the pressure to 0.15 MPa. Start timing for 15 minutes when the temperature reaches 125 °C, and then cool it to room temperature;

[0074] (6) Preparation of date paste

[0075] Wait for the pressure in the pot to drop to normal pressure, take out the jujubes, and use a homogenizer to break and stir them together with the jujube water to obtain jujube paste with a certain water content.

[0076] (7) Preservation

[0077] Place the prepared jujube paste sample in a sterile bag and store it frozen in a -20°C refrigerator.

[0078] Example 3 (comparative example):

[0079] (1) Raw material preparation: Select red jujubes with a round surface, bright color, and the place of origin being Xinjiang. Take them out from the 4°C refrigeration device.

[0080] (2) Soaking and cleaning

[0081] Take the materials, with a material ratio of one part by mass of red jujubes to two parts by mass of water, and soak them to clean the soil and impurities on the surface.

[0082] (3) Drying

[0083] Lay the washed red jujubes flat in a ventilated place to let the water evaporate completely.

[0084] (4) Preparation of jujube paste, without removing the pits

[0085] For the red jujubes without water on the surface, without removing the pits, the mass ratio with water is 1:2.

[0086] (5) Boiling treatment

[0087] Under normal pressure, pour the jujubes and water into the pot and heat. Start timing for 30 minutes when the water boils, and then cool to room temperature.

[0088] (6) Preparation of jujube paste

[0089] Wait for the temperature in the pot to drop to normal temperature, remove the jujube skins and pits, and use a homogenizer to break and stir the jujubes together with the jujube water to obtain jujube paste with a certain water content.

[0090] (7) Preservation

[0091] Place the prepared jujube paste sample in a sterile bag and store it frozen in a -20°C refrigerator.

[0092] Example 4 (comparative example):

[0093] (1) Raw material preparation: Select red jujubes with a round surface, bright color, and the place of origin being Xinjiang. Take them out from the 4°C refrigeration device.

[0094] (2) Soaking and cleaning

[0095] Take the materials, with a material ratio of one part by mass of red jujubes to two parts by mass of water, and soak them to clean the soil and impurities on the surface.

[0096] (3) Air drying

[0097] The washed red dates are laid flat in a ventilated place to evaporate the moisture.

[0098] (4) Preparation of date paste without removing the pits

[0099] The red dates without moisture on the surface, without removing the pits, are in a mass ratio of 1:2 with water.

[0100] (5) Steaming treatment

[0101] Under normal pressure, the dates and water are placed on a steamer tray. Start timing for 30 minutes when the water boils, and then cool to room temperature.

[0102] (6) Preparation of date paste

[0103] When the temperature in the pot drops to normal temperature, remove the date skin and pits, and use a homogenizer to break and stir the dates together with the date water to obtain date paste with a certain water content.

[0104] (7) Preservation

[0105] Place the prepared date paste sample in a sterile bag and store it frozen in a -20°C refrigerator.

[0106] Example 5 (comparative example):

[0107] (1) Raw material preparation: Select red dates with a round surface, bright color, and the place of origin being Xinjiang. Take them out from a 4°C refrigeration device.

[0108] (2) Soaking and cleaning

[0109] Take the materials, with a material ratio of one part of red dates to two parts of water, soak them to clean the soil and impurities on the surface.

[0110] (3) Air drying

[0111] The washed red dates are laid flat in a ventilated place to evaporate the moisture.

[0112] (4) Preparation of date paste without removing the pits

[0113] The red dates without moisture on the surface, without removing the pits, are in a mass ratio of 1:2 with water.

[0114] (5) High-pressure treatment for 30 minutes

[0115] In a high-pressure pot, set the pressure to 0.15 MPa. Start timing for 30 minutes when the temperature reaches 125°C, and then cool to room temperature.

[0116] (6) Preparation of date paste

[0117] Wait for the pressure in the pot to drop to normal pressure, take out the jujubes, remove the jujube skins and pits, and use a homogenizer to break and stir them together with the jujube water to obtain jujube paste with a certain water content.

[0118] (7) Preservation

[0119] Place the prepared jujube paste sample in a sterile bag and store it frozen in a -20°C refrigerator.

[0120] Example 6 (Example):

[0121] (1) Raw material preparation: Select red jujubes with a round surface, bright color, and the place of origin being Xinjiang. Take them out from the 4°C refrigeration device.

[0122] (2) Soaking and cleaning

[0123] Take the materials, with a material ratio of one part by mass of red jujubes to two parts by mass of water, soak them to clean the soil and impurities on the surface.

[0124] (3) Air drying

[0125] Lay the cleaned red jujubes flat in a ventilated place to let the water evaporate completely.

[0126] (4) Air flash evaporation

[0127] For the red jujubes without water on the surface, the mass ratio with water is 1:2.

[0128] When there is a large amount of steam coming out of the steamer, that is, when the temperature in the pot reaches 100°C, place the jujubes and water on the steamer and flash evaporate for 2 minutes, then cool to room temperature of 25°C.

[0129] (5) Ultrasonic light cooking

[0130] For the red jujubes without water on the surface, remove the pits and cut them into pieces, with a mass ratio with water of 1:2.

[0131] Place them in an ultrasonic cleaner for ultrasonic treatment. Pretreat ultrasonically for 40 minutes, remove the pits, at a temperature of 26°C, a power of 60% (rated power 480V), and then cool to room temperature.

[0132] (6) High-pressure steaming for 15 minutes

[0133] In the high-pressure pot, set the pressure to 0.15 MPa. When the temperature reaches 125°C, start timing for 15 minutes, and then cool to room temperature.

[0134] (7) Jujube paste preparation

[0135] Wait for the pressure in the pot to drop to normal pressure, take out the jujubes, and use a homogenizer to break and stir them together with the jujube water to obtain jujube paste with a certain water content.

[0136] (8) Preservation

[0137] Place the prepared jujube paste sample in a sterile bag and store it frozen at -20°C in a refrigerator.

[0138] Note: By measuring the amino acid content of Example 6 (Example), the jujube paste prepared in this example has the highest amino acid content. For this example, peptide screening was carried out, and five new peptide segments, namely Ala-Pro-Ile, Ile-Pro-Phe, Cyclo(Gly-Pro), cyclo(Phe-Ala), and PyroGlu-Pro, were discovered. Functional evaluation of the five newly discovered peptide segments was carried out, and it was found that jujube peptides have preventive and therapeutic effects on Alzheimer's disease (AD), confirming that jujube peptides have neuroprotective functions.

[0139] Example 7 (Comparative Example):

[0140] (1) Raw material preparation: Select red dates with a round surface, bright color, and produced in Xinjiang. Take them out from the 4°C refrigeration device.

[0141] (2) Soaking and cleaning

[0142] Take the material, with a material ratio of one part by mass of red dates to two parts by mass of water, and soak it to clean the soil and impurities on the surface.

[0143] (3) Air drying

[0144] Lay the cleaned red dates flat in a ventilated place to let the water evaporate completely.

[0145] (4) Preparation of jujube paste without removing the pits

[0146] Take the red dates without surface moisture, without removing the pits, with a mass ratio of 1:2 to water.

[0147] (5) High-pressure treatment for 20 min

[0148] In a pressure cooker, set the pressure to 0.15 MPa. Start timing for 20 min when the temperature reaches 125°C, and then cool to room temperature.

[0149] (6) Preparation of jujube paste

[0150] Wait for the pressure in the pot to drop to atmospheric pressure, take out the jujubes, and use a homogenizer to break and stir them together with the jujube water to obtain jujube paste with a certain water content.

[0151] (7) Preservation

[0152] Place the prepared jujube paste sample in a sterile bag and store it frozen at -20°C in a refrigerator.

[0153] Example 8 (Example):

[0154] (1) Raw material preparation: Select red dates from Xinjiang with a round surface, bright color, and take them out from the 4°C refrigeration device;

[0155] (2) Soaking and cleaning

[0156] Take the material, with a material ratio of one part by mass of red dates to two parts by mass of water, soak it, aiming to clean the soil and impurities on the surface;

[0157] (3) Air drying

[0158] Lay the cleaned red dates flat in a ventilated place to evaporate the moisture;

[0159] (4) Air flash evaporation

[0160] For the red dates without moisture on the surface, the mass ratio with water is 1:2;

[0161] When a large amount of steam emerges from the steamer, that is, when the temperature in the steamer reaches 100°C, place the dates and water on the steamer and flash evaporate for 2 minutes, then cool to room temperature;

[0162] (5) Ultrasonic light ripening

[0163] For the red dates without moisture on the surface, remove the pits, cut into pieces, and the mass ratio with water is 1:2;

[0164] Place them in an ultrasonic cleaner for ultrasonic treatment. Pretreat ultrasonically for 30 minutes, remove the pits, at a temperature of 26°C, with a power of 60% (rated power 480V), then cool to room temperature;

[0165] (6) High-pressure treatment for 15 minutes

[0166] In the autoclave, set the pressure to 0.15 MPa. When the temperature reaches 125°C, start timing for 15 minutes, then cool to room temperature;

[0167] (7) Jujube paste preparation

[0168] When the pressure in the pot drops to atmospheric pressure, take out the dates and break and stir them together with the date water using a homogenizer to obtain jujube paste with a certain water content.

[0169] (8) Preservation

[0170] Place the prepared jujube paste sample in a sterile bag and store it frozen in a -20°C refrigerator.

[0171] Example 9 (Example):

[0172] (1) Raw material preparation: Select red dates from Xinjiang with a round surface, bright color, and take them out from the 4°C refrigeration device;

[0173] (2) Soaking and cleaning

[0174] Take the materials, with a material ratio of one part by mass of red dates to two parts by mass of water, and soak them to clean the soil and impurities on the surface;

[0175] (3) Air-dry

[0176] Lay the washed red dates flat in a ventilated place to evaporate the moisture;

[0177] (4) Air flash evaporation

[0178] For the red dates without surface moisture, the mass ratio to water is 1:2;

[0179] When a large amount of steam emerges from the steamer, that is, when the temperature inside the pot reaches 100 °C, place the dates and water on the steamer and flash evaporate for 2 min, then cool to room temperature;

[0180] (5) Ultrasonic light cooking

[0181] For the red dates without surface moisture, remove the pits, cut into pieces, and the mass ratio to water is 1:2;

[0182] Place them in an ultrasonic cleaner for ultrasonic treatment. Pretreat ultrasonically for 20 min, remove the pits, at a temperature of 26 °C, with a power of 60% (rated power 480 V), then cool to room temperature;

[0183] (6) High-pressure treatment for 15 min

[0184] In the autoclave, set the pressure to 0.15 MPa. Start timing for 15 min when the temperature reaches 125 °C, then cool to room temperature;

[0185] (7) Preparation of date paste

[0186] When the pressure inside the pot drops to atmospheric pressure, take out the dates and use a homogenizer to break and stir them together with the date water to obtain date paste with a certain water content.

[0187] (8) Preservation

[0188] Place the prepared date paste sample in a sterile bag and store it frozen in a -20 °C refrigerator.

[0189] Example 10 (Example):

[0190] (1) Raw material preparation: Select red dates with round surfaces, bright colors, and the place of origin being Xinjiang. Take them out from the 4 °C refrigeration device;

[0191] (2) Soaking and cleaning

[0192] Take the materials, with a material ratio of one part by mass of red dates to two parts by mass of water, and soak them to clean the soil and impurities on the surface;

[0193] (3) Air-dry

[0194] The cleaned red dates are laid flat in a ventilated place to evaporate the moisture completely;

[0195] (4) Air flash evaporation

[0196] For the red dates with no moisture on the surface, the mass ratio of red dates to water is 1:2;

[0197] When a large amount of steam emerges from the steamer, that is, when the temperature inside the steamer reaches 100 °C, place the red dates and water on the steamer and flash evaporate for 2 minutes, then cool to room temperature;

[0198] (5) Ultrasonic light ripening

[0199] For the red dates with no moisture on the surface, remove the pits and cut them into pieces. The mass ratio of red dates to water is 1:2;

[0200] Place them in an ultrasonic cleaner for ultrasonic treatment. Conduct ultrasonic pretreatment for 10 minutes, remove the pits, at a temperature of 26 °C and a power of 60% (rated power), then cool to room temperature;

[0201] (6) High-pressure treatment for 15 minutes

[0202] In the autoclave, set the pressure to 0.15 MPa. When the temperature reaches 125 °C, start timing for 15 minutes, then cool to room temperature;

[0203] (7) Preparation of date paste

[0204] Wait until the pressure inside the pot drops to atmospheric pressure, take out the red dates, and use a homogenizer to break and stir them together with the date water to obtain date paste with a certain water content.

[0205] (8) Preservation

[0206] Place the prepared date paste sample in a sterile bag and store it frozen in a -20 °C refrigerator.

[0207] Store the samples prepared in the above examples separately so as to compare the effect examples of date paste obtained by different processes.

[0208] Effect example 1: Determination of amino acid and cyclic adenosine monophosphate contents by liquid chromatography-mass spectrometry

[0209] Determine the amino acids in the date paste obtained from Examples 1-7 with different process differences. Examples 8-10 only have differences in ultrasonic time and are not used for amino acid content determination. Thaw the date paste obtained from Examples 1-7 and conduct high-speed centrifugation at parameters of 1000 r / min for 30 minutes at 4 °C to obtain date paste and date water, and detect the bioactive substances in the date water.

[0210] The detection targets include tryptophan, arginine, histidine, lysine, isoleucine, proline, threonine, valine, alanine, serine, phenylalanine, cysteine, glutamic acid, tyrosine, methionine, glutamine, and aspartic acid. Among them, seven amino acids, namely glutamic acid, methionine, leucine, tyrosine, phenylalanine, lysine, and arginine, are listed as pharmacodynamic amino acids due to their special medical and health care effects in humans and animals. Therefore, the detection of the above amino acids can verify the content of bioactive substances and nutritional value in jujube paste to a certain extent.

[0211] The test results are as Figure 1 shown that the total amino acid and cyclic adenosine monophosphate contents in Example 6 of the present invention are significantly higher than those in the other six groups of examples, and the content of proline is the highest. Therefore, Example 6 is used as the basis.

[0212] Effect Example 2: Liquid chromatography-mass spectrometry - qualitative and quantitative analysis of bioactive substances

[0213] There are significant differences in the content of bioactive substances in jujube paste under different process conditions. Therefore, Examples 1-10 are all measured.

[0214] MS-DIAL was used to rapidly screen for active markers, especially bioactive peptide substances, in Examples 3 and 6 to determine the differences in active ingredients under different processes. MS-DIAL is an analysis software for untargeted metabolomics that can rapidly screen for markers. The software settings are as follows: According to the instrument type, select the data type as "Centroiddata" and the ion mode as positive ion. The liquid chromatography-mass spectrometry data was converted into the.mzML format using the MSConvert software, and the sample files were batch imported into the software, and the corresponding sample types, classification IDs, analysis sequences, injection volumes (μL) information were set. In the data collection tab, set the tolerance of MS1 to 0.01 Da, the tolerance of MS2 to 0.05 Da, set the retention time period to 0-10 min, and the mass number analysis range of MS1 and MS2 to 50-1200 Da. The maximum charge number is 2. Set the minimum peak height to 5000 and the peak width threshold to 0.1. The smoothing method can be used to determine the left and right edges of the peak, and the smoothing levels are set to 3 and the minimum peak width is set to 5 respectively. As shown in Table 1, for the positive ion mode, select the adduct ions as [H] + , [Na] + , [K] + , and [NH4] + .

[0215] Table 1: Specific peptide bioactive substances in high-pressure jujube paste

[0216]

[0217] For the detection items of quercetin, cyclic adenosine monophosphate (cAMP), gallic acid, dihydromyricetin, propyl gallate, cytisine, kojic acid, betulinic acid, and oleanolic acid, all the different processes in Examples 1 - 10 were detected. As Figure 2 shown, among them, the content of the iconic active ingredient cAMP and oleanolic acid detected in jujube was relatively high, and the content determination and comparison were carried out;

[0218] As Figure 2 shown, the results of Effect Example 1 were obtained. The amino acid content in Example 6 was the highest. Combining ultrasonic and high - pressure conditions, in Examples 8 - 10, the changes of bioactive substances under the process conditions of ultrasonic and high - pressure in the examples were comprehensively analyzed. Among them, the content of cAMP and oleanolic acid showed obvious advantages in Example 6, and the content of cAMP was particularly prominent. It was detected by triple quadrupole mass spectrometry to be 370 ± 14 μg / g.

[0219] Effect Example 3: Sensory evaluation

[0220] The sensory evaluation method was used to conduct a sensory assessment of the jujube paste prepared by different processes in Examples 1 - 10. Ten teachers and students with different ages and genders from a food professional background were selected to form a sensory evaluation panel. The entire sensory evaluation process was carried out according to the specific requirements of GB13868 - 2009. According to the analysis method in sensory evaluation, the sensory evaluation rules for jujube paste filling were formulated. Considering the nature of jujube paste itself, whole - wheat sugar - free bread was prepared and the jujube paste was evenly spread to facilitate reasonable evaluation by the evaluators. Each evaluation member conducted a sensory evaluation of the sample's appearance color, flavor, taste, and texture structure one by one by means of visual inspection, nose smelling, and tasting, and the user survey method was used to determine the weights of each item in the sensory determination. The meanings of the test items, their meanings, and their significance for the sensory score of jujube paste are shown in the table. Each evaluation was independently completed by each evaluation member without mutual communication among members. The evaluation weights were: appearance color 20 points, flavor 30 points, taste 30 points, and texture structure 20 points.

[0221] Table 2: Sensory evaluation criteria for jujube paste

[0222]

[0223] The maximum and minimum values in the experimental data were removed, and the data results were expressed in the form of mean ± standard deviation.

[0224] Table 3: Sensory evaluation results of jujube paste

[0225]

[0226] As Figure 3 shown, in summary, based on the total score results of the four evaluation indicators of Examples 1 - 10 by sensory evaluators, asFigure 3 As shown in the figure, the comprehensive score of Example 6 is the highest, and the color, flavor, texture and morphological structure of the jujube paste evaluated by the evaluator are higher than those of other examples.

[0227] Effect Example 4: Electronic nose fingerprint

[0228] The jujube paste obtained from Specific Examples 1-10 was thawed and subjected to high-speed centrifugation at 1000 r / min for 30 min at 4 °C to obtain jujube paste and jujube water, and the bioactive substances in the jujube water were detected.

[0229] The detection object includes the detection and analysis of the electronic nose odor fingerprint.

[0230] The results are as Figure 4 and Figure 5 shown. The results of the detection by the electronic nose principal component analysis method are as Figure 4 shown. The odors of Examples 3 and 4 are significantly different from other conditions; the odors of Examples 2 and 7 are similar and the difference is not significant. The sum of the contribution rates of the first and second principal components in the PCA principal component analysis is close to 97.49%, which basically covers most of the original information of the samples. The contribution rate of the first principal component is 90.15%, that is, the differences between the samples are mainly manifested in the first principal component. Combining with the PCA discrimination analysis table (obtained according to the internal calculation method of the electronic nose software, the closer the value is to 1, the more significant the discrimination of the samples), it can be seen from the PCA analysis that the differences in odor are obvious, while the odors are relatively close under different parameter times and ultrasonic times under high-pressure conditions.

[0231] The Loadings analysis method is related to PCA. They are both based on the same algorithm. However, the difference is that in this experiment, the Loadings algorithm mainly studies the sensors, and this method can be used to confirm the contribution rate of each sensor to the discrimination of the jujube paste samples in different treatment groups of Examples 1-10.

[0232] Table 4: Electronic nose sensor performance table

[0233] Array Serial Number Sensor Name Performance Description 1 W1C Aromatic Components, Benzenes 2 W5S High Sensitivity, Sensitive to Nitrogen Oxides 3 W3C Ammonia Water, Sensitive to Aromatic Components 4 W6S Mainly Selective to Hydrogen 5 W5C Alkane Aromatic Components 6 W1S Sensitive to Short-chain Alkanes such as Methane 7 W1W Sensitive to Inorganic Sulfides 8 W2S Sensitive to Alcohols, Ethers, Aldehydes and Ketones 9 W2W Aromatic Components, Sensitive to Organic Sulfides 10 W3S Sensitive to Alkanes, Long-chain Alkanes

[0234] The results are as Figure 5 shown. In the direction of the first principal component, the contribution rates of sensors No. 1, 8, 6 and 2 are all very large. At the same time, the contribution rates of sensors No. 4 and 6 to the second principal component are also the largest, and sensor No. 7 also makes a certain contribution to the second principal component of the jujube paste;

[0235] Combined with the PCA and loading analysis of the samples, the contribution rate of the first principal component is relatively large, and the differences in the jujube paste samples obtained in Examples 1-10 on the first principal component are also the largest. Therefore, it can be speculated that the differences between the samples are mainly manifested in the aromatic components, alcohol ether aldehyde ketones, and short-chain alkane components such as methane corresponding to sensors No. 1, No. 8, and No. 6.

[0236] Effect Example 5: Headspace solid-phase microextraction-gas chromatography single quadrupole mass spectrometry

[0237] Based on Effect Examples 3 and 4, sensory evaluation and electronic nose were carried out on the jujube paste prepared in Examples 1-10. Through the subjective evaluation and objective analysis of 10 kinds of jujube paste, it was determined that the jujube paste obtained in Example 6 was preferably effective. Therefore, HS-SPEM-GC-MS analysis was carried out on Example 6, as Figure 6 shown.

[0238] Weigh 5 g of jujube paste into a 20 ml headspace vial, then seal it with a cap with a silicone septum, and equilibrate it at 50 °C for 30 min in the heating device of the gas chromatography-mass spectrometry instrument. Then, under the conditions of continuous heating and stirring (300 r / min), insert the extraction head (HP-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm)) into the headspace vial 3 cm above the liquid level of the jujube paste. Insert the extraction head, extract for 30 min, and then insert it into the GC-MS injection port and desorb it at 200 °C for 3 min.

[0239] GC-MS analysis conditions: GC conditions: HP-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm); injection port temperature 200 °C; temperature programming: initial temperature 40 °C, hold for 3 min, rise to 120 °C at 5 °C / min, and then rise to 200 °C at 10 °C / min, hold for 5 min; carrier gas is nitrogen, flow rate is 0.8 mL / min; injection is carried out in splitless mode.

[0240] MS conditions: electron ionization source; electron energy 70 eV; ion source temperature 230 °C; quadrupole temperature 150 °C; transfer line temperature 200 °C; acquisition is in full scan mode, mass acquisition range m / z 33-500. The NIST17.L mass spectrometry database was used for retrieval, and peaks with a matching degree of not less than 80 were selected to determine each flavor substance. Each sample was measured 3 times.

[0241] From Figure 7It can be seen that the volatile aroma components in the treatment group of Example 6 include esters, fatty acids, alkanes, benzenes, alcohols, and alkenes. Among them, the aroma components in the red date puree mainly contribute esters such as dimethylsilanediol, 3-octylcyclopentene, 1,5-cyclooctadiene, decamethylcyclopentasiloxane, and di-n-decyl phosphite, diisobutyl phthalate, 2-(4-morpholinyl)ethyl isothiocyanate, diisooctyl phosphite, and trimethylsilyl ester. It is consistent with the electronic nose analysis results of Effect Example 4, indicating that more aroma component substances are locked in the red date puree prepared in Example 6 of the present invention.

[0242] In summary, the proportion of alcohols in the total aroma flavor substances in Example 6 is relatively large. Among them, the aroma component substances in Example 6 do not contain benzenes and alkenes, indicating that long-term ultrasonic treatment will result in a better enzyme inactivation effect, leading to the decomposition of active ingredient substances to produce more alcohols and reducing the production of benzene and alkene flavor substances during the preparation process.

[0243] Effect Example 6: In vitro antioxidant activity of jujube puree

[0244] Based on the above Effect Examples 1-5 for the comparison of the antioxidant activity of jujube puree, it was determined to select Example 6 and Example 9 of the present invention for comprehensive comparative analysis, and the antioxidant ability of jujube puree was measured respectively for four indexes: DPPH scavenging ability, ABTS + scavenging ability determination, ·OH scavenging ability determination, O2 - scavenging ability determination.

[0245] (1) DPPH scavenging ability determination

[0246] DPPH free radical is a stable free radical centered on nitrogen. Its ethanol solution is purple and has a characteristic absorption at 517 nm. After adding an antioxidant, the antioxidant can combine with or replace the DPPH free radical, reducing the number of free radicals and making the solution color lighter, thereby evaluating its antioxidant ability. First, prepare JAE solutions and dilute them successively with ddH2O into sample solutions with different concentrations (0.05, 0.1, 0.5, 1, 50, 100, 250, 500 mg / ml). Then, prepare VC standard solutions with different concentrations (0.001, 0.0025, 0.01, 0.025, 0.05, 1 mg / ml) of vitamin C reference substance with ddH2O. Weigh 0.79 g of DPPH powder and make it up to 2000 mL with 80% methanol to prepare a 0.91 mM DPPH solution (protected from light). Take a new 96-well plate and set up sample groups, control groups, and blank groups, with three replicates for each concentration in each group (n = 3). For the sample groups, take 40 μL of the sample solution respectively, add 160 μL of 1 mM DPPH solution, incubate at room temperature in the dark for 30 min, and measure the absorbance A1 at 517 nm; use the same method to measure VC Perform a positive control test on the solution.

[0247]

[0248] A0: Replace the JAE sample solution with the same volume of ddH2O; the operation is the same as A1; A2: Replace the DPPH solution with the same volume of 80% methanol; the operation is the same as A1.

[0249] From Figure 8 and Figure 9 it can be seen that as the concentration of the jujube paste supernatant increases, its ability to scavenge DPPH free radicals increases correspondingly. It can be seen that JAE scavenges DPPH free radicals in a dose-dependent manner. The IC50 value of the jujube paste supernatant in Example 6 for scavenging DPPH free radicals is 3.75 mg / mL; the IC50 value of the jujube paste supernatant in Example 9 for scavenging DPPH free radicals is 8.397 mg / mL. It is suggested that at low doses, the DPPH free radical scavenging ability of JAE is still strong, and at the same time, the scavenging ability of JAE reaches 0.95 at 100 mg / mL, and the scavenging ability is at a high stable level. It is suggested that JAE can achieve a strong DPPH free radical scavenging ability at medium and high doses.

[0250] (2) ABTS + Determination of scavenging ability

[0251] ABTS is oxidized to generate a stable green substance ABTS under suitable conditions + , which has a characteristic absorption peak at 734 nm. Adding an antioxidant will inhibit the generation of ABTS + free radicals, weakening the color, thereby evaluating its antioxidant ability

[91] 。First, prepare JAE solutions by sequentially diluting them with ddH2O into sample solutions of different concentrations (0.05, 0.1, 0.5, 1, 50, 100, 250, 500 mg / ml), and prepare VC standard solutions of different concentrations (0.001, 0.0025, 0.01, 0.025, 0.05, 1 mg / ml) by dissolving VC reference substances with ddH2O. Weigh ABTS powder and potassium persulfate powder, and prepare ABTS stock solution (7 mM) and potassium persulfate solution (2.45 mM) with ddH2O for later use. Take appropriate volumes of ABTS stock solution and potassium persulfate solution according to the number of experimental samples, mix them evenly at a ratio of 1:1, react in the dark for 12 - 16 h, and dilute 40 - 50 times with ddH2O before use until the absorbance at 734 nm is 0.7 ± 0.02 to obtain the ABTS working solution. Take a brand - new 96 - well plate and set up sample groups and control groups. Each concentration of samples in each group is set with three replicates (n = 3), and the average value is taken. For the sample group, take 40 μL of the sample solution and 160 μL of the ABTS working solution, mix them evenly by shaking for 10 s, place them at room temperature for 6 min, and measure the absorbance A1 at a wavelength of 734 nm; for the control group, take 40 μL of ddH2O and 160 μL of the ABTS working solution, mix them evenly by shaking for 10 s, place them at room temperature for 6 min, and measure the absorbance A0 at a wavelength of 734 nm. Use the same method to measure the VC solution for positive control experiments.

[0252]

[0253] It can be seen from Figure 10 and Figure 11 that the ability of JAE to scavenge ABTS + radicals is positively correlated with its concentration. The EC 50 value in Example 6 is 2.4183 mg / mL, and the scavenging ability of JAE for ABTS + radicals at a concentration of 50 mg / mL is as high as 0.9. It can be seen that the scavenging ability of JAE for ABTS + radicals is better than that for DPPH radicals, indicating the good antioxidant ability of JAE.

[0254] (3) Determination of ·OH scavenging ability

[0255] Hydroxyl radicals (·OH) react with salicylic acid to form 2,3 - dihydroxybenzoic acid, which has a characteristic absorption peak at 510 nm. Antioxidants can reduce the generation of hydroxyl radicals, resulting in a decrease in absorbance, thereby evaluating their antioxidant ability.

[92] First, prepare JAE solutions by successively diluting them with ddH2O into sample solutions of different concentrations (0.05, 0.1, 0.5, 1, 50, 100, 250, 500 mg / ml), and prepare VC reference solutions of different concentrations (0.001, 0.0025, 0.01, 0.025, 0.05, 1 mg / ml) by dissolving VC reference substances with ddH2O. Use ddH2O to dissolve and continue to prepare 6 mM FeSO4 solution, 6 mM H2O2 solution, and 6 mM salicylic acid (dissolved in absolute ethanol) solution. Take a brand-new 96-well plate and set up sample groups, control groups, and blank groups, with three replicates for each concentration in each group (n = 3). For the sample groups, first take 20 μL of the above-mentioned JAE with different mass concentrations and add them to the corresponding wells, then successively add 20 μL of 6 mM FeSO4 solution and 20 μL of 6 mM H2O2 solution. After standing for 10 min, add 60 μL of 6 mM salicylic acid solution; for the control groups, replace 60 μl of 6 mM salicylic acid solution with 60 μL of absolute ethanol solution, and the other steps are exactly the same as those of the sample groups; for the blank groups, replace 20 μL of JAE with 20 μL of ddH2O solvent, and the other steps are exactly the same as those of the sample groups; after adding all the reagents to each group, incubate in a water bath at 37 °C for 30 min, and measure the absorbances A1 for samples, A2 for controls, and A0 for blanks at 510 nm; use the same method to measure the VC gradient reference substances for positive control experiments.

[0256]

[0257] A0: Replace the JAE solution with the same volume of ddH2O; the operation is the same as A1; A2: Replace the salicylic acid solution with the same volume of absolute ethanol solution; the operation is the same as A1.

[0258] From Figure 12 and Figure 13 , the jujube concentration scavenges superoxide anion O2 - radicals in a dose-dependent manner, which is directly proportional to its concentration. Among them, the ability of jujube to scavenge O2 - radicals is the strongest in Example 6. And the IC - value of the VC reference substance for O2 50 radicals is 1.05723 mg / mL, which is the weakest in the scavenging tests of the four radicals, indicating that the ability of JAE to scavenge O2 - radicals is relatively weak.

[0259] (4) Determination of O2 - scavenging ability

[0260] Pyrogallol undergoes autoxidation reaction to generate superoxide anion (O2 - ) and colored intermediate products. The intermediate products have a characteristic absorption peak at 320 nm, and antioxidants can react with O2 -The reaction prevents the accumulation of intermediate products, weakens the light absorption, and thus evaluates its antioxidant capacity. First, prepare JAE solutions and successively dilute them with ddH2O into sample solutions of different concentrations (0.05, 0.1, 0.5, 1, 50, 100, 250, 500 mg / ml). Prepare VC reference solutions with different concentrations (0.001, 0.0025, 0.01, 0.025, 0.05, 1 mg / ml) by dissolving VC reference substances with ddH2O. Continue to prepare 50 mM Tris-hydrochloric acid buffer solution (PH = 8.2) and 5 mM pyrogallol (dissolved in ddH2O), and preheat them in a water bath at 25 °C for 20 min. Take a 96-well plate, divide it into a sample group, a control group, and a blank group, with three replicates in each group (n = 3), and take their average values. For the sample group, first add 40 μL of JAE solutions of each concentration to each well, then add 120 μL of 50 mM Tris-hydrochloric acid buffer solution, and preheat them in a water bath at 25 °C for 20 min. After the reaction, add 16 μL of 5 mM pyrogallol solution to each well, mix well, and accurately react in a water bath at 25 °C for 5 min (accurately time), and finally add 24 μL of 1.5 M hydrochloric acid to terminate the reaction. Immediately measure the absorbance at a wavelength of 325 nm, which is A1; use the same method to measure the VC gradient reference substances for positive control experiments.

[0261]

[0262] A0: Replace the JAE solution with the same volume of ddH2O; the operation is the same as A1; A2: Replace the pyrogallol solution with the same volume of pyrogallol dilution (ddH2O); the operation is the same as A1;

[0263] From Figure 14 and Figure 15 , the jujube concentration scavenges superoxide anion O2 - free radicals in a dose-dependent manner, which is proportional to its concentration. Among them, the ability of the jujube in Example 6 to scavenge O2 - free radicals is the strongest. And the EC - value of the O2 50 free radical VC reference substance is 1.09712 mg / mL, and its ability is the weakest in the scavenging tests of the four free radicals, indicating that the ability of JAE to scavenge O2 - free radicals is relatively weak.

[0264] In summary, among the antioxidant capacity indicators of the jujube paste prepared in Example 6 and Example 9 of the present invention, the jujube paste prepared in Example 6 shows the strongest antioxidant capacity.

[0265] Effect Example 7: Red date polypeptide - neuroprotective peptide

[0266] Combined with the results of Examples 1-6 of the binding effect, the content of bioactive substances and amino acids in the jujube paste obtained in Example 6 is the highest compared with other examples. The protein concentration of the jujube paste in Example 6 was measured. The protein content in the jujube paste was determined by the Kjeldahl method in accordance with the national food safety standard, Determination of Protein in Foods GB 5009.5-2016. Weigh 2 g to 5 g of the well-mixed semi-solid sample (equivalent to 30 mg to 40 mg of nitrogen), accurate to 0.001 g, and transfer it to a digestion tube. Then add 0.4 g of copper sulfate, 6 g of potassium sulfate and 20 mL of sulfuric acid, and digest it in a digestion furnace. When the temperature of the digestion furnace reaches 420 °C, continue to digest for 1 h. At this time, the liquid in the digestion tube is green and transparent. After taking it out and cooling, add 50 mL of water, and perform the processes of automatic liquid addition, distillation, titration and recording of titration data on an automatic Kjeldahl distiller (add sodium hydroxide solution, hydrochloric acid or sulfuric acid standard solution and boric acid solution containing mixed indicator A or B before use).

[0267] The content of protein in the sample is calculated according to formula (1):

[0268]

[0269] X ——— The content of protein in the sample, unit: grams per hundred grams (g / 100 g);

[0270] V1 ——— The volume of the sulfuric acid or hydrochloric acid standard titrant consumed by the test solution, unit: milliliters (mL);

[0271] V2 ——— The volume of the sulfuric acid or hydrochloric acid standard titrant consumed by the reagent blank, unit: milliliters (mL);

[0272] c ——— The concentration of the sulfuric acid or hydrochloric acid standard titration solution, unit: moles per liter (mol / L);

[0273] 0.0140 ——— The mass of nitrogen equivalent to 1.0 mL of sulfuric acid [c(1 / 2H2SO4) = 1.000 mol / L] or hydrochloric acid [c(HCl) = 1.000 mol / L] standard titration solution, unit: grams (g);

[0274] m ——— The mass of the sample, unit: grams (g);

[0275] V3 ——— The volume of the digested solution absorbed, unit: milliliters (mL);

[0276] F ——— The coefficient for converting nitrogen to protein. The nitrogen conversion coefficients for various foods can be found in Appendix A of GB 5009.5-2016; 100 ——— The conversion coefficient.

[0277] When the protein content is ≥ 1 g / 100 g, the result is retained to three significant figures; when the protein content is < 1 g / 100 g, the result is retained to two significant figures. The protein content of the jujube paste in Example 6 was calculated to be 1.12%.

[0278] For the jujube paste obtained in Example 6, a library search for active ingredients in foodomics was carried out, and peptide segments Ala-Pro-Ile, Ile-Pro-Phe and cyclic dipeptides (Cyclo(Gly-Pro), cyclo(Phe-Ala)) and PyroGlu-Pro were obtained. By comparing the database, these peptide segments were newly discovered, and in vitro cell experiments were carried out on the obtained peptide segments to verify their neuroprotective function.

[0279] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory impairment and cognitive dysfunction, which seriously affects the lives of patients. The supernatant of the jujube paste in Example 6 of the patent was verified for its neuroprotective effect, and the possibility of the extract of the jujube paste to prevent and treat AD in PC12 cells was verified through cell experiments. Virtual screening was carried out to screen peptides with potential memory improvement properties from the jujube paste, and cell experiments were carried out on the screened peptide segments.

[0280] When the PC12 cells reached 90% confluence, a single-cell suspension was prepared. The cells were enumerated under a microscope and then inoculated into a 96-well plate at a density of 1.0×10 5 cells / mL. After inoculation, the cells were cultured for 24 h, and the cells were divided into a blank control group (containing only the basal medium), a control group (the basal medium containing cells), and a sample group (low, medium, and high dose) groups. The blank group (without cells) and the control group were each added with 100 μL of the basal medium. A supernatant of the jujube paste containing 100 μL of the basal medium (concentration gradients of 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL) was established as the experimental group. All cells were cultured for 24 h. Subsequently, 10 μL of CCK-8 solution was added to each well. After the cells were incubated for 1 h, the absorbance was measured at 450 nm, and the cell viability was calculated as follows: Cell viability % = (experimental group - blank group) / (control group - blank group) × 100 (Formula 6)

[0281] The morphology of the cells was observed under a microscope to calculate the cytotoxicity. As Figure 16 , there was no significant difference compared with the control group (p < 0.05), indicating that the jujube supernatant had no obvious toxic effect on the growth and metabolism of the cells. At a concentration of 25 mg / mL, the cell survival rate was 91.88%. This finding provides a safe concentration range for the application of the jujube paste and also verifies the safety of the jujube peptides.

[0282] Detection of cytotoxicity by CCK-8 method

[0283] Four treatment groups were established to explore the effect of jujube puree supernatant on the oxidative stress response of PC12. The specific grouping is as follows: blank group (containing only basal medium), control group (basal medium containing cells), positive VE control group (160 μl of VE added), model group (basal medium treated with H2O2), and sample group (basal medium treated with hydrogen peroxide and polypeptide). After 24 h of cell culture, the basal medium was removed, and 400 μM H2O2 was added for incubation for 24 h to induce oxidative stress. After adding 10 μL of CCK-8 to each group, the cell viability was measured.

[0284] Among them, in the control group and the experimental group, the cell culture medium in each well was removed, 100 μL of PBS was added for washing, and then 100 μL of new complete medium was added to remove the drug effect. In the control group (basal medium containing cells), blank group (containing only basal medium), model group (treated with 400 μM H2O2), and experimental H2O2 groups (low, medium, and high doses), 10 μL of CCK-8 solution was added to each well. After 1 h, the absorbance (OD value) at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated according to the formula:

[0285] Cell viability (%) = [1 - (As - Ab) / (Ac - Ab)] * 100%

[0286] As: Absorbance of the experimental group (containing cells, medium, CCK-8 solution, and drug)

[0287] Ac: Absorbance of the control group (containing cells, medium, CCK-8 solution, without drug)

[0288] Ab: Absorbance of the blank group (containing medium, CCK-8 solution, without cells and drug)

[0289] Such as Figure 17 , cell experiments confirmed that the jujube supernatant can effectively prevent H2O2-induced oxidative stress, and the cell viability was significantly increased, ranging from 41.32% to 72.66%. These findings suggest that jujube puree can be a potential candidate drug for enhancing memory and provide a new approach for the prevention and treatment of AD.

[0290] To determine the sequence of the key peptides with memory-enhancing function in jujube puree. By consulting relevant literature, 67 related neuroprotective peptide segments were sorted out, and LC / MS and virtual screening were used to search for specific peptides with potential neuroprotective function from the jujube puree supernatant. Five new polypeptides, namely Ala-Pro-Ile, Ile-Pro-Phe, cyclo(Gly-Pro), cyclo(Phe-Ala), and PyroGlu-Pro, were screened by molecular docking.

[0291] The cytotoxicity experiment of the discovered polypeptides was carried out by the CCK-8 method. During the cell experiment, the potential of these new red date polypeptides in neuroprotection was demonstrated through the oxidative stress experiment induced by H2O2.

[0292] The cell viability was measured by CCK-8 to judge the protective effect of five liver-protecting peptides on hydrogen peroxide-induced PC12 cell damage. The results are as Figure 18 shown. Compared with the control group, the five peptides had a certain protective effect on hydrogen peroxide-induced PC12 cell damage. Among them, two peptides, Cyclo-GP and Ala-Pro-Ile, showed significant protective effects on cell damage, and the cell survival rates were 88.83% and 78.73% respectively, which were the two peptides with the best effects among the five candidate liver-protecting peptides.

[0293] In summary, Example 6 prepared by the present invention was the best in the effect examples. The red date paste of Example 6 of the invention was verified by in vitro experiments and had neuroprotective function; among them, through virtual screening, separation and purification and other means, five new red date peptides, namely Ala-Pro-Ile, Ile-Pro-Phe, cyclic dipeptide (Cyclo(Gly-Pro)), cyclic dipeptide cyclo(Phe-Ala)) and PyroGlu-Pro, were discovered. The newly discovered red date peptides were verified by in vitro cell experiments, and it was found that two red date peptides, Ala-Pro-Ile and Cyclo-GP, had the best effects in the experiment of inducing cell damage with hydrogen peroxide. Therefore, the red date paste of Example 6 obtained by the present invention was used for intensive processing applications.

[0294] Application Example 1: Red date granules

[0295] For the red date paste prepared in Examples 1-10, according to the results of Effect Examples 1-6, the red date paste of Example 7 was selected to prepare red date paste granules, which provided a certain direction for the application of red date paste. Such as Figure 19 , the red date paste prepared in Example 6 was pretreated by freeze-drying to obtain red date paste freeze-dried powder. The red date paste freeze-dried powder and maltodextrin were mixed at a ratio of 1:0.5. The red date powder contained a high content of polysaccharides and was easy to agglomerate. It was processed by intermittent ultrafine grinding, and the ultrafine ground red date powder and maltodextrin were prepared into granules at -4°C. The red date granules had less water content and were easy to preserve.

[0296] According to the above-prepared red date granules, they had a strong red date aroma, high nutritional value, were easy to preserve, had simple processing conditions, low production equipment costs, and were easy to put into production and use.

[0297] Such as Figure 20As shown, the red date granules can preserve their bioactive components to the greatest extent. Prepared under low-temperature conditions, they will not destroy the original bioactive components of the date paste. The addition of maltodextrin and the lignin of ginseng improves the nutritional value of the granules.

[0298] Application Example 2: Red Date and Collagen Peptide Beverage

[0299] In order to make better use of the produced date paste, a red date and collagen peptide beverage was developed. Using red date juice and milk powder as the main raw materials, red date oligopeptides were added for flavor blending. With the strong sweet flavor of red dates, the common bitter taste of peptides was neutralized while minimizing the types of raw materials. The addition of red date juice gave the product a color different from that of conventional milk beverages. The ingredients were simple and common, reducing the production cost of the milk beverage. Using oligopeptide liquid instead of oligopeptide powder in the formula simplified the formula process and facilitated the industrial production of the product.

[0300] Red date neuroprotective peptide was added to this product as a special nutritional component. It not only enabled the product to have nutritional and health care functions mainly featuring blood nourishment and neuroprotection, but also had the advantage of being more easily digested by the human body compared with the direct application of collagen and plant protein, increasing the value of the product as a health food. It also provided a unique mellow taste to the product. For example Figure 21 and Figure 22 , the date paste obtained in Example 7 was homogenized with water to obtain red date juice. The peptide segments obtained from the date paste in Example 3 and collagen powder were added to the red date juice, and at the same time, milk powder was added to prepare a red date and collagen peptide beverage.

[0301] Using red date juice, milk powder, and red date oligopeptides as the main raw materials, a red date and collagen oligopeptide milk beverage was developed by utilizing the characteristic that oligopeptide products are more easily absorbed by the human body than proteins.

[0302] As can be seen from Examples 1-10 and Effect Examples 1-7, after red dates are subjected to air flash evaporation and ultrasonic light cooking and the high-pressure steaming time is determined to be 15 minutes, a scientific and objective comparison is made with the jujube paste obtained by traditional steaming and boiling methods. The odor and active substance content of the jujube paste are higher than those of other conventional treatment methods. Among them, without adding other chemical agents, the proline, valine, arginine, aspartic acid, asparagine, serine, phenylalanine, tyrosine, and methionine contained in the red date paste prepared in Examples 1-10 have obvious advantages compared with Examples 3 and 4 of the ordinary red date preparation method. Examples 2 and 6 prepared by ultrasonic assistance; cyclic adenosine monophosphate has good sedative, hypnotic and antihypertensive effects, and the effect of improving sleep may also be enhanced in red dates after air flash evaporation, ultrasonic light cooking and high-pressure steaming. The protein content of the jujube paste prepared in Example 6 was measured to be 1.12% by the Kjeldahl method. The supernatant of the jujube paste in Example 6 was extracted and verified by in vitro experiments on PC12 cells. It was found that the cytotoxicity and survival rate of the jujube paste supernatant were not significantly different from those of the control group. By virtual screening of five new polypeptides in red dates with protective effects on AD, namely Ala-Pro-Ile, Ile-Pro-Phe, cyclo(Gly-Pro), cyclo(Phe-Ala), and PyroGlu-Pro, the red date polypeptides screened by virtual screening were synthesized into pure peptide segments with a purity of 98.99%, and the protective effect on hydrogen peroxide-induced PC12 cells was explored. It was found that the extracted peptide segments had a significant protective effect compared with the positive drug VE. Among them, two peptides, Cyclo-GP and Ala-Pro-Ile, showed significant protective effects on hydrogen peroxide-induced damage, and the cell survival rates were 88.83% and 78.73% respectively, which were the two peptides with the best effects among the five candidate liver-protecting peptides. The contents of cAMP and oleanolic acid showed obvious advantages in Example 6, and the cAMP content was as high as 370±14 μg / g; the ultrasonic treatment time had a certain degree of influence on the flavor of the jujube paste, reducing the generation of unpleasant flavor substances, increasing the content of aroma flavor components, and at the same time improving the antioxidant capacity of the jujube paste and effectively increasing the content of active components of the jujube paste. Using Xinjiang red dates as raw materials, through the control of the pressure, time, and temperature of soaking comprehensive pretreatment, air flash evaporation, ultrasonic light cooking, and high-pressure steaming, the prepared jujube paste has high amino acid content, cAMP, and oleanolic acid content, and new neuroprotective peptide sequences are also found. Through Application Examples 1-2, red date granules and red date donkey-hide gelatin peptide beverages are developed, with low production costs, opening up a new way for the application of jujube paste in the health care field, and at the same time providing a new track for the development of Xinjiang red dates.

[0303] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various modifications and refinements can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A preparation method of red date paste with a neuroprotective peptide segment, characterized in that: Proceed as follows S1: Select red dates from Xinjiang with smooth surfaces, bright colors, and the place of origin being Xinjiang. S2: Wash the red dates, dry the washed red dates, then place them in a steam device with a temperature of 95 - 110 °C in a pot for air flash evaporation treatment. After cooling, perform ultrasonic treatment in an ultrasonic cleaner. After ultrasonic treatment, transfer them to a pressure cooker, set the pressure to 0.12 - 0.25 MPa for high-pressure steaming. When the pressure in the pot drops to atmospheric pressure, take out the dates, remove the date skins and pits, and use a homogenizer to break and stir them together with date paste and date paste liquid to obtain red date paste with neuroprotective peptide segments.

2. The preparation method of the red date paste with a neuroprotective peptide segment according to claim 1, characterized in that, In step S1, the variety of the red dates from Xinjiang is Xinjiang Junzao or Xinjiang Huizao.

3. The preparation method of the red date paste with a neuroprotective peptide segment according to claim 1, characterized in that, In step S2, perform air flash evaporation treatment for 1 - 3 min.

4. The preparation method of the red date paste with a neuroprotective peptide segment according to claim 1, characterized in that, In step S2, the conditions for ultrasonic treatment are: set the time to 10 - 40 min and the temperature to 25 - 40 °C.

5. The preparation method of the red date paste with a neuroprotective peptide segment according to claim 1, characterized in that, In step S2, perform high-pressure steaming for 10 - 15 min.

6. The preparation method of the red date paste with a neuroprotective peptide segment according to claim 1, characterized in that, In step S2, set the pressure to 0.15 MPa and perform high-pressure steaming at 125 °C for 15 min.

7. The preparation method of the red date puree with a neuroprotective peptide segment according to claim 1, characterized in that, In step S2, the red date paste with neuroprotective peptide segments contains the following peptide segments: Peptide segment 1: Ala - Pro - Ile Peptide segment 2: Ile - Pro - Phe Peptide segment 3: Cyclo(Gly - Pro) Peptide segment 4: Cyclo(Phe - Ala) Peptide segment 5: PyroGlu - Pro 8. Use of the red date paste with neuroprotective peptide segments prepared by the preparation method according to any one of claims 1 - 6 in the preparation of a red date paste granule product with neuroprotective function.

9. Use of the red date paste with neuroprotective peptide segments prepared by the preparation method according to any one of claims 1 - 6 in the preparation of a red date paste beverage product with neuroprotective function.

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