Application of spirulina phycocyanin peptide in preparation of anti-aging effect product and spirulina phycocyanin peptide beer

Abstract

The invention discloses application of spirulina phycocyanin peptide in preparation of an anti-aging effect product and spirulina phycocyanin peptide beer, and belongs to the technical field of protein peptide application and beer brewing. The spirulina phycocyanin peptide is applied to preparation of an anti-aging effect product, and the amino acid sequence of the spirulina phycocyanin peptide is shown as SEQ ID NO.1. The anti-aging functional product is spirulina phycocyanin peptide beer, spirulina phycocyanin peptide is added 5-8 min before the saccharification step is finished, and the adding amount of the spirulina phycocyanin peptide is 6-8 per thousand. The invention creatively finds that spirulina phycocyanin peptide can promote the synthesis of type I collagen and has an anti-aging effect, the anti-aging effect can be further exerted by preparing the spirulina phycocyanin peptide into functional beer and cooperating with the fermentation process of beer, the application of micromolecule polypeptide in the field of food is expanded, and the application prospect is broad. Good application prospects and excellent economic values are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of protein peptide application and beer brewing technology, and particularly relates to application of a spirulina phycocyanin peptide in preparation of anti-aging efficacy products and spirulina phycocyanin peptide beer. BACKGROUND

[0002] With the improvement of consumer health awareness, traditional industrial beer has been difficult to meet the diversified demand of the market for nutritional and health functions. Functional beer is becoming a new trend in the industry development by virtue of its unique nutritional value and health efficacy. Studies have shown that functional beer not only retains the taste characteristics of traditional beer, but also endows it with health functions such as antioxidant, anti-inflammatory, immune regulation and the like by adding functional ingredients (such as active peptides, plant extracts and the like). The product design combining drinkability and functionality meets the current consumer's pursuit of the concept of "medicinal and edible homology", and has great market potential.

[0003] Spirulina is rich in protein and low in lipid content, and therefore, spirulina protein is a good source of high-quality bioactive peptides. Its various functional properties have been widely studied, including antioxidant, anti-aging, iron chelation, anti-atherosclerosis activity, anti-inflammatory activity, blood pressure-lowering activity and the like. Spirulina phycocyanin peptide is widely used in the fields of drugs, food and cosmetics due to its excellent biocompatibility, easy absorption and safety. However, up to now, few people have applied spirulina-derived polypeptides to beer brewing process. SUMMARY

[0004] In view of the problems in the prior art, the present application provides application of a spirulina phycocyanin peptide in preparation of anti-aging efficacy products and spirulina phycocyanin peptide beer. The spirulina phycocyanin peptide has good anti-aging effect and can be used as an additive component of functional beer, which has good application prospect in the field of beer.

[0005] The present application is achieved by the following technical solutions: In a first aspect, the present application provides application of a spirulina phycocyanin peptide in preparation of anti-aging efficacy products. The amino acid sequence of the spirulina phycocyanin peptide is shown in SEQ ID NO. 1.

[0006] Further, the anti-aging efficacy product is spirulina phycocyanin peptide beer.

[0007] In a second aspect, the present application provides a spirulina phycocyanin peptide beer. The spirulina phycocyanin peptide beer is added with spirulina phycocyanin peptide.

[0008] Further, the preparation process of the spirulina phycocyanin peptide beer comprises the steps of crushing malt, saccharification, filtration, boiling, cyclone sedimentation and fermentation and maturation.

[0009] Further, the spirulina phycocyanin peptide is added 5-8 min before the end of the saccharification step.

[0010] Further, the spirulina phycocyanin peptide is added 5-8 min before the end of the saccharification step.

[0011] Further, the spirulina phycocyanin peptide is extracted from Spirulina platensis or chemically synthesized; through enzyme hydrolysis condition optimization, an enzyme hydrolysis product with higher activity is screened, and the spirulina phycocyanin peptide is separated and purified through ultrafiltration and high performance liquid chromatography (HPLC), and then the structure of the active peptide component is analyzed by LC-MS / MS, and then the physicochemical properties, potential biological activity, toxicity and the like of the polypeptide are predicted by bioinformatics methods, and the polypeptide with biological activity is screened.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application creatively finds that the spirulina phycocyanin peptide (MGHP) can promote the synthesis of type I collagen and has an anti-aging effect; by preparing a functional beer containing the spirulina phycocyanin peptide, the anti-aging effect can be further exerted in cooperation with the fermentation process of the beer, the application of the small molecule polypeptide in the food field is expanded, and the present application has good application prospect and excellent economic value. DETAILED DESCRIPTION

[0013] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods not specified in the following examples are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturers.

[0014] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as familiar to those skilled in the art. The reagents or raw materials used in the present application can be purchased through conventional channels, and the reagents or raw materials used in the present application are used according to the conventional manner or according to the product instructions unless otherwise specified.

[0015] Example 1 The spirulina phycocyanin peptide is obtained according to the method disclosed in patent CN119350478B, and the amino acid sequence (SEQ ID NO. 1) of the spirulina phycocyanin peptide is MGHP (Met-Gly-His-Pro).

[0016] Example 2 The preparation of the spirulina phycocyanin peptide beer with anti-aging effect specifically includes the following steps: (1) Grinding raw materials Add water (20wt%) to the malt, stir thoroughly, and then grind it in a grinder to maximize the crushing of the malt endosperm. The standard for crushing is to break the bran but not crush it. This will form a layer of lees during filtration, which is beneficial for the filtration of wort.

[0017] (2) Saccharification Barley malt and wheat malt were crushed at a ratio of 3:2 (dry weight of malt) and fed into the water at a ratio of 1:3.5. The water temperature was raised to 50°C before feeding. To ensure complete protein decomposition, the temperature was maintained for 30 minutes, and then the temperature was slowly raised to 68°C and maintained for 60 minutes to allow β-amylase to hydrolyze the protein under suitable conditions, which was intended to form fermentable sugars. Finally, the temperature was raised to 78°C. Five minutes before the end of saccharification, 6‰ (dry weight of malt) of spirulina phycocyanin peptide (MGHP) was added. A small amount of wort was then tested for iodine. If there was no obvious discoloration, the saccharification process was considered complete. (3) Filtering After saccharification, the wort is filtered. The wort is then poured into a filter tank and filtered using the filter layer naturally formed by the tank and the wasps. If the filtration process is too slow in the later stages, it can be adjusted by manually stirring (in the bottle) or rotating the tiller (in the pot). When filtration is almost complete, the wasps are washed with 78°C water, and the sugar content of the wort is tested. Throughout the process, the filtered wort must be clear and transparent. Finally, the filtered wort is poured into a boiling kettle.

[0018] (4) Boil After filtering the wort, boil it in a boiling pot and keep it boiling for 70 minutes. Add hops three times during the boiling process, starting from the moment the wort boils. Add 0.1‰ bitter hops after boiling for 10 minutes, add 0.3‰ bitter hops after boiling for 30 minutes, and add 0.2‰ aromatic hops 10 minutes before the end of boiling.

[0019] (5) vortex precipitation After boiling, pour the wort into a vortex settling tank and let it stand for 20 minutes.

[0020] (6) Fermentation and ripening After swirling and settling, the hop mash and hot coagulated material are discharged. The clear wort is cooled via a plate heat exchanger and pumped into the fermentation tank. The inlet temperature is controlled at 18-22°C, and oxygen is introduced to create an environment conducive to yeast initiation and growth. Yeast cells are then inoculated under aseptic conditions. To ensure good yeast growth and metabolic activity, the inoculation concentration is 0.8 × 10⁻⁶. 7After the yeast is inoculated, the wort enters the main fermentation process (the fermentation temperature is controlled at 18-22℃), and the yeast performs aerobic respiration to consume the sugar in the wort during the main fermentation process. Since the yeast is in a vigorous growth stage at this time, the sugar content of the fermentation liquid needs to be detected frequently. When the sugar content is reduced to 4°P, the tank is sealed (the opening degree of the tank top exhaust valve is controlled to keep the tank pressure rising to the required value), and the main fermentation period ends. After the fermentation tank is sealed, the post-fermentation process begins. At this time, the yeast performs anaerobic respiration to generate a large amount of carbon dioxide and ethanol, thus causing the pressure in the fermentation tank to rise. When the pressure in the fermentation tank rises to 0.14 MPa, diacetyl reduction occurs, and the diacetyl reduction is basically completed in about one week. The diacetyl content in the fermentation liquid is detected every day, and when the diacetyl content is less than 0.08 mg / L, the diacetyl reduction process is completed. Then the fermentation tank temperature is reduced to 0℃, and the fermentation tank pressure is controlled at about 0.13 MPa. If the yeast mud at the bottom of the fermentation tank is deposited too much, it needs to be discharged in an appropriate amount, otherwise it will affect the taste of the beer. After the fermentation temperature is reduced to 0℃, it is continued for 7 days to enter the beer maturation period, which aims to stabilize the beer body and observe the pressure in the fermentation tank. In order to ensure that the beer is refreshing and has a good taste, food-grade CO2 gas is introduced in time when the pressure is insufficient.

[0021] Example 3 Compared with Example 2, the addition amount of spirulina phycocyanin peptide (MGHP) in step (2) of Example 3 is 7‰ of the dry weight of malt, and the rest of the operations are the same as those of Example 2.

[0022] Example 4 Compared with Example 2, the addition amount of spirulina phycocyanin peptide (MGHP) in step (2) of Example 4 is 8‰ of the dry weight of malt, and the rest of the operations are the same as those of Example 2.

[0023] Comparative Example 1 Compared with Example 2, the addition amount of spirulina phycocyanin peptide (MGHP) in step (2) of Comparative Example 1 is 2‰ of the dry weight of malt, and the rest of the operations are the same as those of Example 2.

[0024] Comparative Example 2 Compared with Example 2, the addition amount of spirulina phycocyanin peptide (MGHP) in step (2) of Comparative Example 2 is 4‰ of the dry weight of malt, and the rest of the operations are the same as those of Example 2.

[0025] Comparative Example 3 Compared with Example 2, the addition amount of spirulina phycocyanin peptide (MGHP) in step (2) of Comparative Example 3 is 1% of the dry weight of malt, and the rest of the operations are the same as those of Example 2.

[0026] Comparative Example 4 Compared with Example 2, the saccharification process of Step (2) of Comparative Example 4 was not added with spirulina phycocyanin peptide (MGHP), and the rest of the operation was the same as Example 2.

[0027] Test Example 1 Determination of beer physical and chemical indexes: (1) Alcohol content test: according to the volumetric method in the density bottle method in GB / T4928-2008 "Beer Analysis Method".

[0028] (2) Diacetyl content determination: according to the method for measuring diacetyl content in GB / T4928-2008 "Beer Analysis Method"; distill diacetyl out by distillation method, then diacetyl and o-phenylenediamine react to generate 2,3-dimethylquinoxaline, measure the absorbance at wavelength 335 nm, and detect the content of diacetyl.

[0029] (3) Turbidity determination: according to GB / T4928-2008 "Beer Analysis Method", the turbidity of beer was determined by turbidimeter after degassing but not filtering the beer liquid.

[0030] (4) Color determination: according to the EBC colorimeter method in GB / T4928-2008 "Beer Analysis Method", the color of beer was determined. After degassing, the beer liquid was poured into a colorimetric cell, and then compared with a standard color disc. When the color tone was consistent, the color of the beer liquid was read out, with unit EBC.

[0031] (5) Foam determination: the fineness, color and hanging cup degree of foam were observed by eyes.

[0032] (6) Foam retention determination: the beer sample was placed in a water bath at about 20℃, and the temperature was kept for 30 min. The foam retention cup was cleaned thoroughly and ready for use. Then, the stopwatch method in GB / T4928-2008 "Beer Analysis Method" was used for detection.

[0033] (7) Beer pH value determination: the beaker and electrode were cleaned with distilled water, then the water on the electrode was absorbed with absorbent paper, and the pH meter was calibrated with standard solution. After calibration, the electrode was cleaned with distilled water again, the distilled water on the electrode was wiped dry, and the electrode was inserted into the measured liquid. When the data showed stable, the reading was directly taken.

[0034] (8) Beer total acid determination First, prepare a standard sodium hydroxide solution (0.1 mol / L) and a phenolphthalein reagent (5 g / L). In a conical flask, add 100 mL of distilled water and boil for 2 min. Then add 10 mL of the sample, heat for another 1 min, turn off the heat, let stand for 5 min, rinse the flask with tap water, add 0.5 mL of phenolphthalein reagent, and titrate with 0.1 mol / L sodium hydroxide solution until a pale pink color is achieved. Record the volume of sodium hydroxide solution consumed. Total acid = 10 × C × V; C – Concentration of sodium hydroxide solution, mol / L; V – The volume of sodium hydroxide solution consumed, in mL.

[0035] (9) The physicochemical properties of the beers prepared in Examples 2-4 and Comparative Examples 1-4 were tested, and the results are shown in Table 1 below: Table 1 Comparison of Physicochemical Indicators of Beer As shown in Table 1, the physicochemical properties of beer are directly related to the entire brewing process and the amount of spirulina protein peptides added. The beer foam is rich, white and delicate, with good cling to the glass. The alcohol content, diacetyl content and total acid content all meet the national standard requirements. The alcohol content of the beer in Examples 2-4 is higher than that of Comparative Examples 1-4, and the foam retention of Examples 3 and 4 is better.

[0036] Experimental Example 2 Beer sensory evaluation After the beer stabilized, a sensory evaluation was conducted on the finished beer by a 10-member judging panel (personnel trained in beer tasting from the School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences)). The beer was comprehensively evaluated based on color, clarity, foam, aroma, and taste. The detailed sensory evaluation scoring criteria are shown in Table 2, with a maximum score of 100 points, and the average score was taken. The beer sensory evaluation table is shown in Table 3. Table 2 Sensory Evaluation Scoring Rules Table 3. Sensory Evaluation Table for Beer As shown in Table 3, the beers prepared in Examples 2-4 scored higher, at 93, 93, and 94 respectively. They were clear, bright in color, with fine white foam, harmonious aroma, rich fragrance, full-bodied and mellow taste, and a refreshing seaweed aroma. Comparative Examples 1-4 scored lower than Examples 2-4 in terms of aroma, taste, body richness, foam, and clarity.

[0037] Experimental Example 3 Experiment to promote the synthesis of type I collagen (collagen I) The experiment was performed using human skin fibroblasts (HSF) as a model cell line.

[0038] 1) Cell inoculation: human skin fibroblasts (HSF) were inoculated into a 24-well plate at a seeding density of 4 x 10 4 cells / well, and incubated in an incubator (37°C, 5% CO2) overnight; 2) Solution preparation: solutions of the samples to be tested were prepared according to the test scheme table (Table 4); 3) Dosing: according to the test scheme in Table 4, when the cell plating rate in the 24-well plate reached 40-60%, the samples to be tested were dosed in groups (2 mL per well, with 3 replicates in each group); after dosing, the 6-well plate was placed in an incubator (37°C, 5% CO2) for 24 h; 4) UVA irradiation: according to the test groups, the groups that needed UVA irradiation were irradiated with 30 J / cm 2 of UVA, and then placed in an incubator (37°C, 5% CO2) for 24 h; 5) Sample collection: after 24 h of incubation, the cell culture supernatant was collected in an EP tube and stored in a -80°C freezer.

[0039] 6) ELISA detection: detection was performed according to the operating instructions of the ELISA kit; 7) Statistical analysis of results: the results were plotted using GraphPad Prism, and the results were expressed as the mean value. The t-test was used for statistical analysis between groups. 0.01 p <0.05 was considered to be significantly different, p <0.01 was considered to be extremely significantly different.

[0040] Table 4 Test scheme table .

[0041] The results of the collagen I content detection are shown in Table 5. As can be seen from Table 5, compared with the BC group, the collagen I content of the NC group decreased significantly, indicating that the test stimulation conditions were effective. Compared with the NC group, the collagen I content of the PC group increased significantly, indicating that the positive control test was effective. Compared with the NC group, Examples 2-4 and Comparative Examples 1-4 all had the effect of promoting the content of collagen I, and Examples 2-4 significantly increased the content of collagen I, with an increase rate of 56.49%, 67.43%, and 73.69%, respectively. The promoting effect of Examples 2-4 was significantly higher than that of Comparative Examples 1-4, among which Example 4 had the highest increase rate, so the beer can significantly increase the content of collagen I and achieve the effect of tightening.

[0042] Table 5 Summary of collagen I content detection results (Note: ## indicates compared with blank control, p <0.01; ** indicates compared with negative control, p <0.01; * indicates compared with negative control, 0.01 p <0.05).

[0043] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application has been particularly described with reference to the examples given, the ordinary skilled person can modify or equivalently replace the technical solutions of the present application according to the needs without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of a Spirulina platensis phycocyanin peptide for the preparation of an anti-aging efficacy product, characterized in that, The amino acid sequence of the spirulina phycocyanin peptide is shown as SEQ ID NO.

1.

2. The use of the peptide of the phycocyanin of the spirulina according to claim 1 for the preparation of an anti-aging efficacy product, characterized in that, The anti-aging functional product is spirulina phycocyanin peptide beer.

3. A spirulina phycocyanin peptide beer, characterized in that, The spirulina phycocyanin peptide of claim 1 or 2 is added.

4. The Spirulina phycocyanin peptide beer according to claim 3, characterized in that, The preparation process of the spirulina phycocyanin peptide beer comprises the steps of malt crushing, saccharification, filtration, boiling, cyclone sedimentation and fermentation after-ripening.

5. The Spirulina phycocyanin peptide beer according to claim 4, characterized in that, The spirulina phycocyanin peptide is added 5-8 minutes before the end of the saccharification step.

6. The Spirulina phycocyanin peptide beer according to claim 5, characterized in that, The added amount of the spirulina phycocyanin peptide is 6-8 ‰ of the dry weight of the malt.

7. The Spirulina phycocyanin peptide beer according to claim 3, characterized in that, The spirulina phycocyanin peptide is extracted from spirulina platensis or chemically synthesized.

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