Hydrolyzed aloe flower protein, preparation method thereof and application thereof in preparing anti-glycosylation products

Abstract

The present application relates to a kind of hydrolyzed aloe flower protein and its preparation method and application in the preparation of anti-glycosylation product, belong to biotechnology field.The present application provides a kind of aloe flower peptide, the aloe flower peptide includes the characteristic peptide of at least one of amino acid sequence Leu-Pro-Phe, Leu-Gly and Ala-Phe.The present application is obtained by treating aloe flower to obtain aloe flower peptide, and the characteristic peptide containing amino acid sequence Leu-Pro-Phe, Leu-Gly and Ala-Phe in aloe flower peptide is identified by HPLC-MS / MS.The present application is proved by experiment that aloe flower peptide, hydrolyzed aloe flower protein containing aloe flower peptide all have the effect of improving AGEs inhibition rate, indicate that it has anti-glycosylation activity, can protect protein substrate from glycosylation modification, and have the advantages such as safety, long-term use, no side effect, can be applied to anti-glycosylation and its related symptom product.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a hydrolyzed aloe flower protein, a preparation method thereof, and an application thereof in the preparation of an anti-glycosylation product. Background Art

[0002] Aloe vera, also known as oil shallot, is a perennial evergreen herb in the genus Aloe in the Liliaceae family. There are numerous species of aloe vera, with nearly 400 known species and over 500 varieties. Aloe vera is primarily native to the Mediterranean coast and the arid tropical regions of southern Africa. It is also found in southern Asia, including India and Malaysia. Aloe vera has numerous medicinal benefits, such as promoting wound healing, combating ulcers, and promoting laxative effects.

[0003] In recent years, as people have deepened their research on aloe vera, the biological activity and material basis of aloe vera have been continuously revealed. Among the applications of aloe vera industry, aloe vera cosmetics have the most developed varieties, the best market acceptance and the greatest added value. As a natural moisturizer and skin nutrient, it can enhance skin metabolism, replenish skin moisture, prevent skin keratinization and maintain skin elasticity. It also has the functions of nourishing hair, killing bacteria and relieving itching, preventing hair from drying out and removing dandruff. At present, whitening creams, sunscreens, anti-wrinkle creams and facial masks made with aloe vera as raw material use the natural moisturizing properties of aloe vera gel to maintain skin elasticity and radiance, soften cuticles, soothe the skin and prevent skin photoaging, meeting the market demand for healthy and harmless skin care cosmetics with beauty and health functions, showing its unique industrial development advantages.

[0004] Although aloe vera and its extracts have been studied relatively comprehensively, traditional extraction processes are unable to obtain the protein substances in aloe vera flowers, and the potential active protein sequences contained therein have not yet been identified and explored. Therefore, there is an urgent need to further research and develop the protein components in aloe vera flowers and their extract residues to improve the resource utilization of aloe vera flowers. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a hydrolyzed aloe flower protein and a preparation method thereof and an application thereof in the preparation of anti-glycation products.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a hydrolyzed aloe flower protein comprising a characteristic peptide having an amino acid sequence of at least one of Leu-Pro-Phe, Leu-Gly and Ala-Phe.

[0008] The hydrolyzed aloe flower protein is obtained by treating aloe flowers, and the characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Gly and Ala-Phe are identified in the hydrolyzed aloe flower protein by HPLC-MS / MS.

[0009] As a preferred embodiment of the hydrolyzed aloe flower protein, the characteristic peptide with the amino acid sequence of Leu-Pro-Phe, the characteristic peptide with the amino acid sequence of Leu-Gly and the characteristic peptide with the amino acid sequence of Ala-Phe are contained.

[0010] Advanced glycation end products (AGEs) are compounds formed by non-enzymatic reactions between sugars and biological molecules such as proteins, lipids or nucleic acids. These products are usually accumulated under hyperglycemic conditions and are closely related to aging and the development of various chronic diseases. AGEs not only affect protein structure and function, but also trigger inflammatory responses by activating receptors, accelerating tissue damage and disease progression. Therefore, reducing the formation of AGEs is of great significance for maintaining health and preventing chronic diseases. The present application proves by experiments that aloe flower peptides and hydrolyzed aloe flower proteins containing aloe flower peptides both have the effect of improving the AGEs inhibition rate, indicating that they both have anti-glycation activity, can protect protein substrates from glycation modification, and have the advantages of safety, long-term use and no side effects, and can be applied to products for anti-glycation and related symptoms.

[0011] As a preferred embodiment of the hydrolyzed aloe flower protein, the mass ratio of the characteristic peptide Leu-Pro-Phe, the characteristic peptide Leu-Gly and the characteristic peptide Ala-Phe in the hydrolyzed aloe flower protein is Leu-Pro-Phe: Leu-Gly: Ala-Phe = (93.0-95.3): (1.0-1.7): (3.0-6.0).

[0012] In a second aspect, the present application provides a preparation method of the above-mentioned hydrolyzed aloe flower protein, comprising the following steps:

[0013] (1) mixing aloe flower powder with water, adjusting pH to 10.0-11.0, extracting 3 times, combining the extraction solutions, and centrifuging to obtain supernatant;

[0014] (2) adjusting the supernatant obtained in step (1) to pH 2.5-3.5, standing, and centrifuging to obtain a precipitate;

[0015] (3) dissolving the precipitate obtained in step (2) in water, adjusting pH to 6.0-8.0, adding protease, water-bathing at 45-55℃ for 4-6h, and centrifuging to obtain an enzymatic hydrolysate;

[0016] (4) The enzymatic hydrolyzate obtained in step (3) is ultrafiltered and dried to obtain hydrolyzed aloe flower protein.

[0017] This invention extracts aloe flower protein from aloe flowers using an alkali-acid precipitation method, then subjecting it to enzymatic hydrolysis, ultrafiltration, and drying to produce hydrolyzed aloe flower protein containing aloe flower peptides. In vivo and in vitro experiments have demonstrated that the hydrolyzed aloe flower protein exhibits excellent anti-glycation efficacy. Different enzymatic hydrolysis conditions (temperature, pH, and hydrolysis time) affect product formation and, in turn, the activity of the hydrolyzed product. Experiments have confirmed that the preferred enzymatic hydrolysis temperature is 45-55°C, with an optimal temperature of 50°C; the enzymatic hydrolysis pH is 6.0-8.0, with an optimal pH of 7.0; and the enzymatic hydrolysis time is 4-8 hours, with an optimal hydrolysis time of 4 hours.

[0018] As a preferred embodiment of the preparation method of the present invention, it includes at least one of the following (I) to (II):

[0019] (I) In step (1), the ratio of aloe pollen to water is aloe pollen: water = 1 g: (10-15) mL;

[0020] (II) In step (1), the extraction is performed in a water bath at 37°C for 40-60 minutes.

[0021] As a preferred embodiment of the preparation method of the present invention, it includes at least one of the following (III) to (IV):

[0022] (III) In step (3), the type of protease includes at least one of papain, trypsin, flavor protease and pepsin;

[0023] (IV) In step (3), the amount of protease added is 5500-6000 U of protease per 1 g of precipitate.

[0024] Generally, different proteases produce different hydrolysis products when hydrolyzing the same substrate. Based on this, the present invention experimentally evaluated the differences in the anti-glycation efficacy of hydrolyzed aloe flower protein prepared using different types of proteases. The results showed that hydrolyzed aloe flower protein prepared using papain, trypsin, flavor protease, and pepsin exhibited superior anti-glycation activity. Therefore, papain, trypsin, flavor protease, and pepsin can be selected as proteases to prepare hydrolyzed aloe flower protein. Furthermore, the proteases used in the enzymatic hydrolysis process of the preparation method of the present invention can be a combination of multiple proteases, such as a combination of papain and trypsin, a combination of papain and flavor protease, or a combination of papain and pepsin.

[0025] As a preferred embodiment of the preparation method of the present invention, in step (4), the ultrafiltration is to place the enzymatic hydrolyzate obtained in step (3) in an ultrafiltration membrane for ultrafiltration separation, and the cut-off molecular weight of the ultrafiltration membrane is 2500-3000Da.

[0026] The present invention has found through experiments that compared with unhydrolyzed aloe flower protein, the anti-glycosylation activity of hydrolyzed aloe flower protein is significantly increased, and the anti-glycosylation activity of hydrolyzed aloe flower protein with a molecular weight of less than 3000Da is even better, which is related to the reduced molecular weight or the release of specific amino acid sequences.

[0027] As a preferred embodiment of the preparation method of the present invention, in step (1), the aloe pollen is prepared by naturally air-drying the aloe flowers, crushing them and passing them through a 40-mesh sieve to obtain the aloe pollen.

[0028] As a preferred embodiment of the preparation method of the present invention, in step (1), the raw materials of the aloe pollen include whole aloe flowers and residues produced by aloe flowers used in the preparation of essences, essential oils or extracts.

[0029] In a third aspect, the present invention provides an aloe flower peptide, which is composed of a characteristic peptide having an amino acid sequence of at least one of Leu-Pro-Phe, Leu-Gly and Ala-Phe.

[0030] In a fourth aspect, the present invention provides the use of the above-mentioned aloe flower peptide or hydrolyzed aloe flower protein in the preparation of anti-glycation products.

[0031] The aloe flower peptides and hydrolyzed aloe flower protein provided by the present invention not only inhibit AGE formation but also significantly suppress AGE-induced inflammatory responses, significantly reducing the release of AGE-induced inflammatory mediators, such as TNF-α and IL-6, thereby alleviating inflammatory damage to tissues and organs. Therefore, the aloe flower peptides and hydrolyzed aloe flower protein provided by the present invention have the dual efficacy of inhibiting AGE formation and AGE-induced inflammation, offering significant advantages among anti-glycation products.

[0032] As a preferred embodiment of the application of the present invention, the products include medicines, cosmetics and biological products.

[0033] As a preferred embodiment of the application of the present invention, the dosage of the hydrolyzed aloe flower protein is 250-1000 mg per 1 kg body weight of the organism.

[0034] In a fourth aspect, the present invention provides an anti-glycation cosmetic comprising the above-mentioned aloe flower peptide or hydrolyzed aloe flower protein.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention obtains hydrolyzed aloe flower protein by processing aloe flowers, and identifies the aloe flower peptides as containing characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Gly and Ala-Phe by HPLC-MS / MS.

[0037] 2. The present invention has experimentally confirmed that aloe flower peptides and hydrolyzed aloe flower proteins containing aloe flower peptides have the effect of increasing the AGEs inhibition rate, indicating that they both have anti-glycation activity, can protect protein substrates from glycation modification, and have the advantages of being safe, long-term use, and having no side effects. They can be used in products for anti-glycation and its related symptoms.

[0038] 3. The present invention extracts aloe flower protein from aloe flowers by using an alkali dissolution and acid precipitation method, and then performs proteolysis, ultrafiltration and drying on the aloe flower protein to prepare hydrolyzed aloe flower protein containing aloe flower peptides. In vivo and in vitro experiments have verified that the hydrolyzed aloe flower protein has excellent anti-glycation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the liquid-to-water BPC graph of hydrolyzed aloe flower protein in Example 1 of the present invention;

[0040] Figure 2 This is the secondary mass spectrum of the LPF peptide segment in Example 1 of the present invention;

[0041] Figure 3 This is the secondary mass spectrum of the LG peptide segment in Example 1 of the present invention;

[0042] Figure 4 This is the secondary mass spectrum of the AF peptide segment in Example 1 of the present invention. DETAILED DESCRIPTION

[0043] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0044] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.

[0045] There is no limitation on the species of aloe used in the aloe flower peptide and hydrolyzed aloe flower protein of the present invention. The aloe used in the following examples and comparative examples is Aloe vera.

[0046] The ultrafiltration membrane was purchased from Suez Environment Group and was made of TFM with a molecular weight cut-off of 3000Da.

[0047] Human fibroblasts were purchased from the China Science Cell Bank with the catalog number GNHu49. The cell culture conditions were based on the description of human fibroblasts in the China Science Cell Bank.

[0048] The solid-phase synthesis of LPF, LG, and AF characteristic peptides is commissioned to a biological company. The commissioned biological company only needs to have the qualification to synthesize peptides, and there are no restrictions.

[0049] SPF female BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center (SCXK (Yue) 2022-0002).

[0050] SPF-grade Kunming female mice were purchased from Guangdong Medical Laboratory Animal Center (SCXK (Yue) 2022-0002).

[0051] The ELISA kit for AGEs was purchased from Beijing Myrida Technology Co., Ltd. with the catalog number M150216-96T.

[0052] The IL-6 ELISA kit was purchased from Beijing Myrida Technology Co., Ltd. with the catalog number MF01310-96T.

[0053] The TNF-α ELISA kit was purchased from Beijing Mairuida Technology Co., Ltd. with the catalog number MF02810-48T.

[0054] The techniques not described in detail in the following examples, comparative examples and effect examples are all commonly used techniques in the art. Please refer to "Molecular Biology Experiment Manual" (Ma Wenli, People's Military Medical Press), "Molecular Biology Experiment (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiment" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0055] In the following examples and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine ​​(Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), and "T" refers to threonine (Thr).

[0056] In the following examples, comparative examples and effect examples, LPF refers to the Leu-Pro-Phe characteristic peptide, LG refers to the Leu-Gly characteristic peptide, and AF refers to the Ala-Phe characteristic peptide.

[0057] Example 1

[0058] This embodiment provides a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method comprising the following steps:

[0059] S1. Aloe vera flowers are naturally air-dried and crushed through a 40-mesh sieve to obtain aloe vera pollen;

[0060] S2. Take the aloe pollen obtained in step S1 and mix it with water according to the ratio of aloe pollen: water = 1 g: 10 mL, adjust the pH to 11.0 with 2M NaOH solution, extract in a 37°C water bath for 40 min, repeat the extraction three times, combine the three extracts, and centrifuge at 10,000 rpm for 10 min to obtain a supernatant;

[0061] S3. The supernatant obtained in step S2 was adjusted to pH 3.0 using 2M HCl solution, allowed to stand for precipitation, and centrifuged at 10,000 rpm for 10 min to obtain a precipitate, which is the crude protein;

[0062] S4. Add water to the precipitate obtained in step S3, add 10 mL of water for every 1 g of precipitate, stir evenly, adjust the pH to 7.0 with 2 M NaOH solution, place in a water bath at 50°C, add 6000 U of papain for every 1 g of precipitate, stir at 300 rpm for 4 h, place in a water bath at 90°C for 15 min, cool to room temperature, centrifuge at 10000 rpm for 10 min, collect the supernatant, and the obtained supernatant is the enzymatic hydrolyzate;

[0063] S5. The enzymatic hydrolyzate obtained in step S4 is ultrafiltered using a laboratory membrane separation device (Hefei Woteng Membrane Separation Equipment Co., Ltd.). The ultrafiltration membrane used has a molecular weight cutoff of 3000 Da to obtain an ultrafiltration solution with a molecular weight less than 3000 Da. The ultrafiltration solution is spray-dried in a high-speed centrifugal spray dryer to obtain hydrolyzed aloe flower protein, which contains aloe flower peptides.

[0064] Example 2-3

[0065] Examples 2-3 respectively provide a hydrolyzed aloe flower protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that:

[0066] Example 2: In step S4, the step of "placing in a water bath at 50°C" is adjusted to "placing in a water bath at 45°C", and the other operations remain unchanged;

[0067] Example 3: In step S4, the “placed in a water bath at 50° C.” is adjusted to “placed in a water bath at 55° C.”, and the other operations remain unchanged.

[0068] Examples 4-5

[0069] Example 4-5 respectively provides a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method is similar to example 1, and the difference is as follows:

[0070] Example 4: in step S4, the "adjusting pH=7.0 with 2M NaOH solution" is adjusted to "adjusting pH=6.0 with 2M NaOH solution", and the rest remains unchanged;

[0071] Example 5: in step S4, the "adjusting pH=7.0 with 2M NaOH solution" is adjusted to "adjusting pH=8.0 with 2M NaOH solution".

[0072] Example 6-9

[0073] Example 6-9 respectively provides a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method is similar to example 1, and the difference is as follows:

[0074] Example 6: in step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 5h", and the rest remains unchanged;

[0075] Example 7: in step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 6h", and the rest remains unchanged;

[0076] Example 8: in step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 7h", and the rest remains unchanged;

[0077] Example 9: in step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 8h", and the rest remains unchanged.

[0078] Example 10-12

[0079] Example 10-12 respectively provides a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method is similar to example 1, and the difference is as follows:

[0080] Example 10: in step S4, the "6000U papain is added to each 1g precipitate" is adjusted to "6000U trypsin is added to each 1g precipitate", and the rest remains unchanged;

[0081] Example 11: in step S4, the "6000U papain is added to each 1g precipitate" is adjusted to "6000U flavor protease is added to each 1g precipitate", and the rest remains unchanged;

[0082] Example 12: In step S4, the step of “adding 6000 U of papain per 1 g of precipitate” is adjusted to “adding 6000 U of pepsin per 1 g of precipitate”, and the other operations remain unchanged.

[0083] Comparative Examples 1-4

[0084] Comparative Examples 1-4 respectively provide a hydrolyzed aloe flower protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that:

[0085] Comparative Example 1: In step S4, the step of “adding 6000 U / g of papain” was adjusted to “adding 6000 U / g of alkaline protease”, and the other operations remained unchanged;

[0086] Comparative Example 2: In step S4, the phrase "add 6000 U of papain per 1 g of precipitate" was adjusted to "add 6000 U of neutral protease per 1 g of precipitate", and the remaining operations remained unchanged;

[0087] Comparative Example 3: In step S4, the step of “adding 6000 U of papain per 1 g of precipitate” was adjusted to “adding 6000 U of composite protease per 1 g of precipitate”, and the remaining operations remained unchanged;

[0088] Comparative Example 4: In step S4, the step of “adding 6000 U of papain per 1 g of precipitate” was adjusted to “adding 6000 U of bromelain per 1 g of precipitate”, and the other operations remained unchanged.

[0089] Comparative Examples 5-8

[0090] Comparative Examples 5-8 respectively provide a hydrolyzed aloe flower protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that:

[0091] Comparative Example 5: In step S4, the step of “placing in a water bath at 50°C” was changed to “placing in a water bath at 30°C”, and the remaining operations remained unchanged;

[0092] Comparative Example 6: In step S4, the step of “placing in a water bath at 50°C” was changed to “placing in a water bath at 35°C”, and the remaining operations remained unchanged;

[0093] Comparative Example 7: In step S4, the step of “placing in a water bath at 50°C” was changed to “placing in a water bath at 40°C”, and the remaining operations remained unchanged;

[0094] Comparative Example 8: In step S4, the “placing in a water bath at 50° C.” is adjusted to “placing in a water bath at 60° C.”, and the other operations remain unchanged.

[0095] Comparative Examples 9-11

[0096] Comparative Example 9-11 respectively provide a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method is similar to that of Example 1, the difference is as follows:

[0097] Comparative Example 9: In step S4, the "adjusting pH=7.0 with 2M NaOH solution" is adjusted to "adjusting pH=5.0 with 2M NaOH solution", and the rest of the operation is unchanged;

[0098] Comparative Example 10: In step S4, the "adjusting pH=7.0 with 2M NaOH solution" is adjusted to "adjusting pH=9.0 with 2M NaOH solution";

[0099] Comparative Example 11: In step S4, the "adjusting pH=7.0 with 2M NaOH solution" is adjusted to "adjusting pH=10.0 with 2M NaOH solution".

[0100] Comparative Example 12-14

[0101] Comparative Example 12-14 respectively provide a hydrolyzed aloe flower protein and a preparation method thereof, the preparation method is similar to that of Example 1, the difference is as follows:

[0102] Comparative Example 12: In step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 1h", and the rest of the operation is unchanged;

[0103] Comparative Example 13: In step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 2h", and the rest of the operation is unchanged;

[0104] Comparative Example 14: In step S4, the "300rpm stirring enzyme hydrolysis for 4h" is adjusted to "300rpm stirring enzyme hydrolysis for 3h", and the rest of the operation is unchanged.

[0105] Comparative Example 15

[0106] Comparative Example 15 provides a hydrolyzed aloe leaf protein and a preparation method thereof, the preparation method is similar to that of Example 1, the difference is as follows:

[0107] In step S1, the "aloe flower" is replaced with "aloe leaf";

[0108] In step S5, the spray drying process is adjusted to freeze drying, and the rest of the steps and parameter conditions are unchanged.

[0109] Example 1

[0110] The hydrolyzed aloe flower protein obtained in Example 1 and the hydrolyzed aloe leaf protein obtained in Comparative Example 15 are characterized, and the specific scheme is as follows:

[0111] 1. Determination of amino acid composition in hydrolyzed aloe flower protein and hydrolyzed aloe leaf protein.

[0112] According to the safety national standard GB 5009.124-2016 Determination of Amino Acids in Foods, the amino acid content of the hydrolyzed aloe flower protein prepared in Example 1 was analyzed using an S7130 amino acid automatic analyzer (SYKAM, Germany), and the separation column was Cation Separation Column LCA K06 / Na, 150 mm x 4.6 mm; the ammonia filtration column was Ammonia Filtration Column LCA K04 / Na, 100 mm x 4.6 mm, and the determination results are shown in Table 1.

[0113] Table 1 Amino acid content of hydrolyzed aloe flower protein and hydrolyzed aloe leaf protein

[0114]

[0115] As shown in Table 1, glutamic acid, serine and the like are the main amino acids of the hydrolyzed aloe flower protein, followed by aspartic acid and leucine, and the content of acidic amino acids of the aloe vera flower extract protein is relatively high, which indicates that the aloe vera flower protein is acidic under natural conditions, which is consistent with the isoelectric point results. The amino acid composition of the hydrolyzed aloe leaf protein is different from that of the hydrolyzed aloe flower protein, such as the percentage of serine and glycine being lower than that in the hydrolyzed aloe flower protein, and the percentage of arginine and tyrosine being higher than that in the hydrolyzed aloe flower protein, indicating that the proteins extracted from different parts of aloe have certain differences.

[0116] 2. Identification of the peptide sequence of the hydrolyzed aloe flower protein obtained in Example 1.

[0117] The amino acid sequence of the hydrolyzed aloe flower protein prepared in Example 1 was identified by HPLC-MS / MS method, and the specific detection method was as follows:

[0118] An X500 LC-ESI-Q-TOF high-resolution liquid chromatograph-mass spectrometer (AB SCIEX, USA) was used for separation and detection of the sample. The liquid chromatograph and mass spectrometer control software was SCIEX OS 2.0 (AB SCIEX, USA); the chromatographic column was 1 x 100 mm HSS T3 (1.8 μm, 100 Å, Waters, USA).

[0119] The mobile phase consists of 0.1% (v / v) formic acid aqueous solution (A) and acetonitrile (B), and the elution method is as follows: 0-4.00 min 5.0% B, 4.00-6.00 min 5.0-10.0% B, 6.00-30.00 min 10.0-40.0% B, 30.00-34.00 min 40.0-90.0% B, 34.00-40.00 min 90% B, 40.00-42.00 min 90.0-5.0% B, 42.00-52.00 min 5.0% B, the flow rate is 0.05 mL / min, the injection amount is 1 μL, the column temperature is 40°C, and the percentage of the above mobile phase means volume percentage, and the volume of the mobile phase B is the total volume of the mobile phase A and the mobile phase B.

[0120] Mass spectrometry detection method: scan period 0.642 s, ESI ion source temperature 500°C, positive ion mode, spray voltage 5500V, TOF primary scan range 100-1200Da, secondary scan range 50-1200Da, working mode IDA, maximum candidate ion number 4, dynamic exclusion is turned on, and the rest of the parameters use the default values of the proteomics method. Note that before using the instrument, the dead volume thereof should be minimized, and the identification results are shown in Table 1. Figure 1-4 and Table 2.

[0121] Table 2 Identification results of aloe flower peptide sequences in hydrolyzed aloe flower protein

[0122]

[0123] As Figure 1-4 As shown in Table 1 and Table 2, three characteristic amino acid sequences are identified from the hydrolyzed aloe flower protein by using HPLC-MS / MS technology, including two dipeptides (LG, AF) and one tripeptide (LPF).

[0124] Effect Example 2

[0125] The hydrolyzed aloe flower protein obtained in Example 1 is subjected to anti-glycosylation (AGEs) activity evaluation, and the specific scheme is as follows:

[0126] In this effect example, the crude protein obtained in step S3 of Example 1, the enzyme hydrolysate obtained in step S4 of Example 1, the components of >3000 Da and <3000 Da obtained in step S5 of Example 1, and the LPF, LG and AF characteristic peptides synthesized by solid phase are selected for experiments.

[0127] A reaction solution containing bovine serum albumin (BSA, 2.8 mg / mL) and methylglyoxal (1 mmol / L) was prepared in PBS. One mL of the reaction solution and one mL of the sample solution (2 mg / mL for aloe vera flower protein, enzymatic hydrolysate, fractions less than 3000 Da, and fractions greater than 3000 Da, respectively; 0.5 mM for LPF, LG, and AF, respectively) were thoroughly mixed in a glass test tube. The tube was sealed with parafilm and placed in a constant temperature incubator at 37°C for 7 days to react and form the glycosylated albumin complex (AGEs-BSA).

[0128] A blank control group was prepared by mixing 1 mL of the reaction solution with 1 mL of PBS. AGEs-BSA exhibits characteristic fluorescence absorption properties (excitation wavelength 370 nm, emission wavelength 450 nm). Fluorescence spectroscopy was used to determine the level of AGEs-BSA production, and the following formula was used to calculate the levels. The results are shown in Table 3:

[0129] AGEs-BSA inhibition rate (%) = [1- (A sample / A blank)] × 100%

[0130] Wherein, A sample: the absorbance value of the sample, A blank: the absorbance value of the blank control.

[0131] Table 3 AGEs-BSA inhibition rate in different treatment groups

[0132]

[0133] As shown in Table 3, while crude aloe flower protein itself exhibits some anti-glycation activity, its activity is significantly enhanced after enzymatic hydrolysis, with the AGEs-BSA inhibition rate increasing from 15.6% to 31.2%. The AGEs-BSA inhibition rate of hydrolyzed aloe flower protein with a molecular weight of less than 3000 Da reaches over 50%. This indicates that the present invention, through enzymatic hydrolysis and ultrafiltration, obtains hydrolyzed aloe flower protein containing low molecular weight, significantly improving the performance and functional value of aloe flower protein. The three characteristic peptides (LPF, LG, and AF) in the hydrolyzed aloe flower protein also exhibit potent anti-glycation activity, with AGEs-BSA inhibition rates exceeding 40%. Compared to aloe flower, aloe leaf protein and its enzymatic hydrolyzate exhibit lower AGEs-BSA inhibition rates, all below 10%. The AGEs-BSA inhibition rate of its fraction less than 3000 Da is only 11.9%, indicating that its efficacy is far inferior to that of the hydrolyzed aloe flower protein of the present invention.

[0134] Effect Example 3

[0135] The anti-glycation activity of the hydrolyzed aloe flower proteins obtained in Examples 1-10 and Comparative Examples 1-14 was tested, and the AGEs-BSA inhibition rate was tested according to the method of Effect Example 2. The results are shown in Table 4.

[0136] Table 4 Effects of different enzymatic hydrolysis processes on the anti-glycosylation activity of hydrolyzed aloe flower protein

[0137]

[0138] As shown in Table 4, the anti-glycation activity of hydrolyzed aloe flower proteins prepared using different processes varied significantly. The anti-glycation activity of hydrolyzed aloe flower proteins prepared using different proteases varied significantly, with the following order of activity, from highest to lowest: papain (52.1%), flavor protease (40.5%), trypsin (36.1%), pepsin (35.2%), composite protease (33.4%), neutral protease (28.9%), bromelain (25.3%), and alkaline protease (19.2%). Therefore, papain is preferred for preparing the anti-glycation aloe flower peptides in the present invention.

[0139] In addition to proteases, the enzymatic hydrolysis temperature, pH value, and enzymatic hydrolysis time also have a certain impact on the anti-glycation activity of hydrolyzed aloe flower protein. Through experiments, it was found that the anti-glycation activity of the product was better when the enzymatic hydrolysis temperature was controlled within the range of 45-55℃, with 50℃ being the optimal temperature; the anti-glycation activity of the product was better when the enzymatic hydrolysis pH was within the range of 6.0-8.0, with 7.0 being the optimal temperature; and the anti-glycation activity of the product was better when the enzymatic hydrolysis time was within the range of 4-8h, with 4h being the optimal temperature.

[0140] Effect Example 4

[0141] The effect of the hydrolyzed aloe flower protein obtained in Example 1 on AGEs in the skin tissue of a skin aging mouse model was tested. The specific protocol is as follows:

[0142] Six-week-old SPF-grade female BALB / c mice (18-22 g) were acclimated for one week in an environment with an ambient temperature of 20-25°C, 40%-60% humidity, and 12 hours of light per day (7:00-19:00). The experiment was then conducted. All mice were randomly divided into a normal control group, a D-galactose model group, a low-dose hydrolyzed aloe flower protein (250 mg / kg) group, a medium-dose hydrolyzed aloe flower protein (500 mg / kg) group, and a high-dose hydrolyzed aloe flower protein (1000 mg / kg) group, with eight mice per group. Except for the normal control group, mice in the remaining groups received a subacute skin aging model via subcutaneous injection of D-galactose (500 mg / kg) at the nape of the neck, once daily for eight consecutive weeks. Starting from the day of model establishment, mice in the treatment group received daily oral administration of the hydrolyzed aloe flower protein prepared in Example 1, while mice in the normal control and model groups received an equal volume of normal saline. After the experiment, the mice were killed by cervical dislocation, the back hair was cut, the back skin was collected, the subcutaneous tissue was removed, and the skin was stored in a -80℃ refrigerator.

[0143] The mouse skin tissue was cut into small pieces, and a proper amount of liquid nitrogen was added to harden the tissue. The tissue was ground with a mortar. RIPA protein lysis buffer was added, and the homogenate was homogenized for 2 min using an automatic homogenizer. The lysis was continued on ice for 30 min. After centrifugation at 12,000 r / min at 4°C for 10 min, the supernatant was collected, and the content of AGEs was detected by using an ELISA kit.

[0144] Table 5: Levels of AGEs in the skin tissues of aging mice in different treatment groups

[0145]

[0146] As shown in Table 5, compared with the normal control group of mice, the level of AGEs in the skin tissues of the D-galactose skin aging model group of mice was significantly increased, indicating a higher glycosylation modification level in the skin tissues. The aloe flower peptide can significantly reduce the content of AGEs in the skin of aging mice, which indicates that the aloe flower peptide provided by the present application can inhibit the formation of AGEs in vivo and reduce the glycosylation modification and functional damage of biological molecules in the skin tissues.

[0147] Effect Example 5

[0148] The effect of the hydrolyzed aloe flower protein obtained in Example 1 on the AGEs in the tissues and organs of the diabetic mouse model was detected, and the specific scheme was as follows:

[0149] The 6-week-old SPF Kunming female mice (18-22 g) were adaptively fed for one week in an environment with an ambient temperature of 20-25°C, a humidity of 40%-60%, and 12 hours of light per day (7:00-19:00). After fasting for 12 hours, the mice were intraperitoneally injected with a 1wt% streptozotocin (STZ) solution (40 mg / kg) for 5 consecutive days. During the modeling period, 10wt% sucrose was added to the drinking water. Three weeks after the first injection of STZ, the fasting blood glucose of the mice was measured. When the fasting blood glucose was higher than 11.1 mmol / L, it was considered that the hyperglycemia model was successfully modeled.

[0150] The mice were randomly divided into a normal control group, a model control group, a low-dose (250 mg / kg) hydrolyzed aloe flower protein group, a medium-dose (500 mg / kg) hydrolyzed aloe flower protein group, and a high-dose (1000 mg / kg) hydrolyzed aloe flower protein group, with 8 mice in each group. Except for the normal control group, the mice in the other groups were given the hydrolyzed aloe flower protein by gavage once a day for 6 consecutive weeks. The normal control group and the model group were given an equal volume of normal saline. After the experiment, the mice were sacrificed by decapitation, the back hair was cut off, the back skin was collected, the subcutaneous tissue was removed, and the eyeball, kidney, heart, liver, and other tissues were collected and stored in a-80°C refrigerator. The levels of AGEs in the tissues of the mice were detected according to the method described in Effect Example 4, and the results are shown in Table 6.

[0151] Table 6 Levels of AGEs in various organs of diabetic mice

[0152]

[0153] As shown in Table 6, compared with the normal control group mice, the levels of AGEs in the skin tissue, eyes, kidneys, heart, liver and other tissues of the model control group mice were significantly increased, while the hydrolyzed aloe flower protein could significantly reduce the AGEs in these tissues and organs. These results suggest that the hydrolyzed aloe flower protein provided by the present invention can alleviate the glycated damage to multiple tissues and organs of the body under high sugar environment, and is expected to prevent and treat various diabetic complications.

[0154] Effect Example 6

[0155] AGEs not only affect the structure and function of proteins by cross-linking and damaging them, but can also activate inflammatory responses. These two combined effects significantly harm the body. Studies have shown that AGEs activate multiple inflammatory signaling pathways by binding to their receptors, such as RAGE, leading to the release of inflammatory mediators, which in turn damage the health of cells and tissues. Therefore, this study evaluated the effect of the hydrolyzed aloe flower protein obtained in Example 1 on AGE-induced inflammatory responses. The specific scheme is as follows:

[0156] Human fibroblasts were plated in a 96-well plate. After the cells adhered overnight, AGEs and the hydrolyzed aloe flower protein obtained in Example 1 were simultaneously administered to the cells at three final concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. After 48 hours, the culture supernatant was collected and the levels of IL-6 and TNF-α were detected by ELISA. The results are shown in Table 7.

[0157] Table 7 Levels of inflammatory factors in the supernatants of cell culture treated with different methods

[0158]

[0159] As shown in Table 7, compared with the blank control group, AGEs can significantly promote the release of IL-6 and TNF-α from human skin fibroblasts, while hydrolyzed aloe flower protein can significantly reduce the level of inflammatory factors, thereby eliminating further tissue damage caused by AGEs through inflammation induction.

[0160] In summary, the hydrolyzed aloe flower protein provided by the present invention has dual effects, which can not only inhibit the formation of AGEs, but also alleviate the inflammatory storm induced by AGEs, reflecting comprehensive anti-glycation effects.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing hydrolyzed aloe flower protein, characterized in that: The following steps are involved: (1) Aloe vera pollen was mixed with water, the pH was adjusted to 10.0-11.0, and the extracts were combined and centrifuged to obtain the supernatant; (2) The supernatant obtained in step (1) was adjusted to pH = 3.0, allowed to stand, and centrifuged to obtain a precipitate; (3) Dissolve the precipitate obtained in step (2) in water, adjust the pH to 6.0-8.0, add papain, incubate in a water bath at 45-55°C for 4-6 hours, and centrifuge to obtain an enzymatic solution; (4) ultrafiltration of the enzymatic hydrolyzate obtained in step (3) and drying to obtain hydrolyzed aloe flower protein; In step (1), the ratio of aloe pollen to water is aloe pollen: water = 1 g: (10-15) mL, and the extraction is performed in a water bath at 37° C. for 40-60 min; In step (4), the ultrafiltration is to place the enzymatic hydrolysate obtained in step (3) in an ultrafiltration membrane for ultrafiltration separation, and the cut-off molecular weight of the ultrafiltration membrane is 3000Da; In step (1), the aloe pollen is obtained by naturally air-drying aloe flowers, crushing them and then passing them through a 40-mesh sieve.

2. The preparation method according to claim 1, wherein In step (3), the amount of protease added is 5500-6000 U of protease per 1 g of precipitation.

3. The preparation method according to claim 1, wherein The hydrolyzed aloe flower protein contains characteristic peptides with amino acid sequences of Leu-Pro-Phe, Leu-Gly and Ala-Phe.

4. Use of hydrolyzed aloe flower protein in the preparation of an anti-glycation product, wherein the hydrolyzed aloe flower protein is obtained by the preparation method according to any one of claims 1 to 3, and the product is selected from cosmetics or medicines.

Images