Preparation method of psidium guajava young fruit extract, product and application thereof

Abstract

The present application relates to a preparation method of psidium guajava young fruit extract and its product and application, the preparation method comprises: crushing psidium guajava young fruit and mixing with water, using microjet extraction to obtain the first mixture; the first mixture is mixed with composite enzyme and enzymolysis, then short time treatment in high temperature pressure environment, obtain the second mixture; the second mixture is filtered by ceramic membrane, reduced pressure concentration, sterilization and drying, then get it. The extract obtained by the preparation method has excellent antioxidant, anti-inflammatory, anti-aging and anti-glycation effects, and good application, and is suitable for various preparation types. Compared with the traditional process, the present application can maximize the production efficiency and product yield, reduce the production cost, and enhance the economic benefit. In addition, the method does not use organic reagent, energy saving and environmental protection, simple operation, suitable for industrial production, and provides an efficient solution for food and skin care products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and food science, and relates to a preparation method of Psidium guajava immature fruit extract, products thereof and applications. BACKGROUND

[0002] Psidium guajava L. is an evergreen shrub or small tree of the Myrtaceae family and Psidium genus, which is originally from the tropical regions of South America and is now widely distributed in southern China, such as Guangdong, Fujian and Yunnan provinces. Recent studies have shown that Psidium guajava has multiple physiological effects, such as reducing blood sugar, lowering blood pressure and anti-virus. The immature fruit and leaves of Psidium guajava have been included in the Guangdong Province Chinese Herbal Medicine Standard (First Edition), and the main effects include astringing intestine, stopping diarrhea, hemostasis, and are used for treating diarrhea and unceasing diarrhea. The immature fruit of Psidium guajava has the effect of reducing blood sugar.

[0003] Although the research on mature fruit and leaves of Psidium guajava is relatively sufficient, the research on its immature fruit is still very limited. Psidium guajava immature fruit is usually considered as a byproduct during planting, however, compared with mature fruit, the immature fruit shows stronger biological activity and higher medicinal value. Therefore, it is particularly important to develop specific extraction technology for Psidium guajava immature fruit.

[0004] The commonly used extraction technologies for Psidium guajava include decoction, organic solvent extraction and fermentation extraction. The decoction method is simple to operate, but has low extraction efficiency, high energy consumption, and long time high temperature treatment may cause loss of active ingredients. The organic solvent extraction has high extraction efficiency, but uses a large amount of organic solvent, which increases the cost and brings potential environmental pollution, and the residual organic solvent may also affect the safety of the product, limiting its application in food and health products. The fermentation extraction has certain advantages, but requires specific high-activity strains, and the production process is easily affected by environmental changes, which increases the cost and complexity.

[0005] Therefore, it is of great significance to develop a new type of extraction method with low energy consumption, simple process, environmental protection and high efficiency for realizing the industrialized production of Psidium guajava immature fruit and making full use of it. This new method should be able to effectively extract and retain more active ingredients, while reducing energy consumption and environmental impact, meet the high standards of modern food and health food industry, simplify the operation process, and ensure the quality stability of the extract, and be easy to scale up. SUMMARY

[0006] In view of the limitations of the prior art, the purpose of the present application is to provide a preparation method of Psidium guajava immature fruit extract, products thereof and applications.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a preparation method of Psidium fructus young fruit extract, the preparation method comprising:

[0009] (1) crushing Psidium fructus young fruit, mixing with water, and then performing micro-jet extraction to obtain a first mixture;

[0010] (2) mixing the first mixture with a compound enzyme and performing enzymolysis, and then incubating to obtain a second mixture;

[0011] (3) filtering the second mixture through a ceramic membrane, concentrating under reduced pressure, sterilizing, and drying to obtain the Psidium fructus young fruit extract.

[0012] The present application overcomes the deficiencies of traditional single process in terms of low raw material yield, severe loss of nutritional components, and complex composition by combining modern biotechnology and physical treatment technology. The preparation method adopted is not only simple and efficient, but also sustainable, suitable for large-scale industrial production, and significantly improves the raw material yield and extraction efficiency, reduces the production cost. At the same time, this method can effectively retain and improve active substances such as Psidium fructus polyphenols and polysaccharides, and enhance the biological activity. The obtained extract exhibits excellent antioxidant, anti-inflammatory, anti-aging and anti-glycation effects, providing high-nutrition-value components for the food and health product industries. In addition, the method of the present application does not use organic solvents in the production process, ensuring environmental protection and low energy consumption, and is a sustainable solution.

[0013] Preferably, the crushed Psidium fructus young fruit further comprises sieving.

[0014] Preferably, the temperature of the micro-jet extraction is 30-60 ℃, and the number of times is 1-6.

[0015] The temperature can be selected from 30 ℃, 32 ℃, 35 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 48 ℃, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃, etc., and the number of times can be selected from 1, 2, 3, 4, 5, 6, etc. Other specific point values within the above numerical range can also be selected, which will not be described here.

[0016] The pressure of the micro-jet extraction is 100-250 Mpa, and the mass ratio of material to liquid is 1:(10-40).

[0017] The pressure can be selected as 100 MPa, 110 MPa, 120 MPa, 130 MPa, 140 MPa, 150 MPa, 160 MPa, 170 MPa, 180 MPa, 190 MPa, 200 MPa, 210 MPa, 220 MPa, 230 MPa, 240 MPa, 250 MPa, etc., and specific point values in (10-40) can be selected as 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0018] Preferably, the complex enzyme is a combination of neutral protease, xylanase, pectinase and tannase.

[0019] The application creatively finds that the combination of neutral protease, xylanase, pectinase and tannase has a certain synergistic effect on the enzymatic effect.

[0020] Preferably, the mass ratio of the neutral protease, xylanase, pectinase and tannase is (2-3):(2-3):(1-2):(1-2).

[0021] Specific point values in (2-3) can be selected as 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc., and specific point values in (1-2) can be selected as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0022] Preferably, the mass of the complex enzyme is 2-3% (for example, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, etc.) of the mass of the guavas, the temperature of the enzymolysis is 40-50 ℃ (for example, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, etc.), and the time is 30-40 min (for example, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, etc.).

[0023] Other specific point values in the above numerical range can be selected, which will not be repeated here.

[0024] Preferably, the holding pressure is 0.1-0.2 MPa, the temperature is 120-130 ℃, and the time is 10-20 min.

[0025] The pressure can be selected as 0.1 MPa, 0.11 MPa, 0.12 MPa, 0.13 MPa, 0.14 MPa, 0.15 MPa, 0.16 MPa, 0.17 MPa, 0.18 MPa, 0.19 MPa, 0.2 MPa, etc., the temperature can be selected as 120 ℃, 121 ℃, 122 ℃, 123 ℃, 124 ℃, 125 ℃, 126 ℃, 127 ℃, 128 ℃, 129 ℃, 130 ℃, etc., and the time can be selected as 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0026] Preferably, the pore size of the ceramic membrane is 100-220 nm, and the temperature of the second mixture when filtered through the ceramic membrane is 30-40 ℃.

[0027] The pore size of the ceramic membrane can be selected as 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, etc., and the temperature can be selected as 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, etc., and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0028] In a second aspect, the present application provides a Psidium fructus extract prepared by the preparation method of the Psidium fructus extract according to the first aspect.

[0029] In a third aspect, the present application provides an application of the Psidium fructus extract according to the second aspect in the preparation of a product with anti-inflammatory, anti-aging, and anti-glycation effects.

[0030] Preferably, the product includes health products or cosmetics.

[0031] Preferably, the dosage form of the product includes powder, granules, hard capsules, soft capsules, tablets, soft candy, solution, jelly, emulsion.

[0032] Preferably, the cosmetic includes a mask, a cream, an essence, a lotion, a toner, or a color cosmetic.

[0033] Preferably, the Psidium fructus extract is added to the product in an amount of 0.2-20%, such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, etc., and other specific point values within the above numerical range can be selected, which will not be repeated here.

[0034] Preferably, the product further comprises excipients and / or additives.

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

[0036] (1) The present application successfully obtains a Psidium fructus extract by combining microfluidic enzymolysis, short-time high-temperature high-pressure treatment and membrane filtration technology for the first time.

[0037] (2) The preparation method of the present application significantly improves the yield of the finished product, and the yield of the extract after drying is more than 35%. The polyphenol content is more than 5%, and the polysaccharide content is more than 13%, showing a high nutritional value.

[0038] (3) After treatment under specific temperature and membrane pore size conditions, the Psidium fructus extract has significantly improved antioxidant, anti-inflammatory, anti-aging and anti-glycation biological activities.

[0039] (4) The extract obtained by the preparation method of the present application has an IC50 value of less than 70 μg / mL in scavenging DPPH free radicals, and can significantly inhibit collagenase and hyaluronidase activity and inhibit the generation of AGEs.

[0040] (5) The extract obtained by the preparation method of the present application can significantly inhibit the secretion of NO and the expression of TNF-α and IL-6 in macrophages, and reduce the level of reactive oxygen species ROS in HaCaT cells. It can also significantly increase the collagen content of zebrafish and significantly inhibit the aggregation of neutrophils in the zebrafish skin inflammation model.

[0041] (6) The extract obtained by the preparation method of the present application has strong antioxidant, anti-inflammatory, anti-aging and anti-glycation activities, and can be used as a natural active ingredient in health products.

[0042] (7) The extract powder obtained by the preparation method of the present application has good flowability, and the NTU value of a 5% concentration aqueous solution is less than 10, with high clarity and stable properties, no visible precipitate or flocculent material, and is suitable for various types of preparations, including powders, granules, liquid preparations, etc.

[0043] (8) The present application combines modern biotechnology and physical treatment technology, does not use organic reagents throughout, not only improves the extraction efficiency and biological activity of guava young fruit extract, but also expands its application range in health products, has significant market potential and application value. The method has strong coherence, short extraction time, energy saving and environmental protection, simple operation, and is beneficial to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a real photo of guava young fruit extract.

[0045] Figure 2 is a HaCaT cell ROS removal effect result graph.

[0046] Figure 3 is a macrophage RAW264.7 NO content result graph.

[0047] Figure 4 is a macrophage RAW264.7 IL-6 gene relative expression amount result graph.

[0048] Figure 5 is a macrophage RAW264.7 TNF-α gene relative expression amount result graph.

[0049] Figure 6 is a zebrafish collagen content result graph.

[0050] Figure 7 is a zebrafish neutrophil number result graph.

[0051] Figure 8 is a zebrafish neutrophil fluorescence staining graph. DETAILED DESCRIPTION

[0052] The technical solutions of the present application will be further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations of the present application.

[0053] The efficacy ingredients in the products involved in the following examples and comparative examples are derived as follows (only the efficacy ingredients are embodied, and the necessary auxiliary ingredients contained in other commercially available raw materials are not described again):

[0054] The neutral protease is a product with the trade name of neutral protease purchased from Henan Wanbang Chemical Technology Co., Ltd.;

[0055] The xylanase is a product with the trade name of xylanase purchased from Shandong Longkete Enzyme Preparation Co., Ltd.;

[0056] The pectinase is a product with the trade name of pectinase purchased from Henan Junyi Biological Technology Co., Ltd.;

[0057] Tannase is a product with the trade name of Tannase purchased from Henan Junyi Biotechnology Co., Ltd.

[0058] Example 1

[0059] The present example provides a preparation method of Psidium fructus extract, which comprises the following steps:

[0060] (1) The Psidium fructus is crushed, passed through a 200-mesh screen, and subjected to micro-jet extraction (temperature is 50 ℃, pressure is 200 Mpa, solid-liquid mass ratio is 1:20, and cycle number is 3 times);

[0061] (2) A compound enzyme (the compound enzyme comprises neutral protease, xylanase, pectinase, and tannase at a mass ratio of 2:2:1:1) is added to the product obtained in step (1), the addition amount of the compound enzyme is 2.5% of the mass of the Psidium fructus, the enzymolysis temperature is 45 ℃, the enzymolysis time is 35 min, then the temperature is kept at 125 ℃ and 0.15 MPa for 15 min;

[0062] (3) After the temperature is reduced to 35 ℃, the product obtained in step (2) is passed through a 400-mesh screen, a 1-μm filter bag, and a 140-nm ceramic membrane, vacuum reduced pressure concentration, UHT ultra-high temperature instantaneous sterilization, freeze-drying, crushing and sieving, and the Psidium fructus extract is obtained.

[0063] Example 2

[0064] The present example provides a preparation method of Psidium fructus extract, which comprises the following steps:

[0065] (1) The Psidium fructus is crushed, passed through a 200-mesh screen, and subjected to micro-jet extraction (temperature is 60 ℃, pressure is 100 Mpa, solid-liquid mass ratio is 1:10, and cycle number is 2 times);

[0066] (2) A compound enzyme (the compound enzyme comprises neutral protease, xylanase, pectinase, and tannase at a mass ratio of 1:1:1:1) is added to the product obtained in step (1), the addition amount of the compound enzyme is 3% of the mass of the Psidium fructus, the enzymolysis temperature is 50 ℃, the enzymolysis time is 30 min, then the temperature is kept at 130 ℃ and 0.2 MPa for 10 min;

[0067] (3) After the temperature is reduced to 30 ℃, the product obtained in step (2) is passed through a 400-mesh screen, a 1-μm filter bag, and a 100-nm ceramic membrane, vacuum reduced pressure concentration, UHT ultra-high temperature instantaneous sterilization, freeze-drying, crushing and sieving, and the Psidium fructus extract is obtained.

[0068] Example 3

[0069] The present example provides a preparation method of Psidium fructus extract, which comprises the following steps:

[0070] (1) The guava young fruit is crushed and passed through a 200-mesh screen, and is subjected to microfluidization (temperature 30°C, pressure 250 Mpa, solid-liquid ratio 1:40, and 6 cycles);

[0071] (2) The product obtained in step (1) is added with a compound enzyme (the compound enzyme includes neutral protease, xylanase, pectinase, and tannase at a mass ratio of 3:3:1:1), and the addition amount of the compound enzyme is 2% of the mass of the guava young fruit, and after enzymolysis at 40°C for 40 min, the product is kept at 120°C and 0.1 MPa for 20 min;

[0072] (3) After the temperature is reduced to 40°C, the product obtained in step (2) is passed through a 400-mesh screen, a 1-μm filter bag, and a 220-nm ceramic membrane, is subjected to vacuum and reduced-pressure concentration, UHT ultra-high-temperature instantaneous sterilization, freeze-drying, and crushing and sieving, and thus the guava young fruit extract is obtained.

[0073] Example 4

[0074] The present example provides a preparation method of a guava young fruit extract, which is different from example 1 only in that step (2) is “the product obtained in step (1) is added with a compound enzyme (the compound enzyme includes neutral protease, xylanase, and pectinase at a mass ratio of 2:2:1), and the addition amount of the compound enzyme is 2.5% of the mass of the guava young fruit, and after enzymolysis at 45°C for 35 min, the product is kept at 125°C and 0.15 MPa for 15 min”, and other operations remain unchanged.

[0075] Example 5

[0076] The present example provides a preparation method of a guava young fruit extract, which is different from example 1 only in that step (2) is “the product obtained in step (1) is added with a compound enzyme (the compound enzyme includes xylanase, pectinase, and tannase at a mass ratio of 2:1:1), and the addition amount of the compound enzyme is 2.5% of the mass of the guava young fruit, and after enzymolysis at 45°C for 35 min, the product is kept at 125°C and 0.15 MPa for 15 min”, and other operations remain unchanged.

[0077] Example 6

[0078] The present example provides a preparation method of a guava young fruit extract, which is different from example 1 only in that step (2) is “the product obtained in step (1) is added with a compound enzyme (the compound enzyme includes neutral protease, pectinase, and tannase at a mass ratio of 2:1:1), and the addition amount of the compound enzyme is 2.5% of the mass of the guava young fruit, and after enzymolysis at 45°C for 35 min, the product is kept at 125°C and 0.15 MPa for 15 min”, and other operations remain unchanged.

[0079] Example 7

[0080] The present example provides a preparation method of Psidium fructus extract, which is only different from example 1 in that step (2) is "adding a compound enzyme (the compound enzyme includes neutral protease, xylanase, tannase in a mass ratio of 2:2:1) to the product obtained in step (1), the addition amount of the compound enzyme is 2.5% of the mass of Psidium fructus, the enzymolysis temperature is 45 ℃, the enzymolysis time is 35 min, then keeping at 125 ℃, 0.15 MPa for 15 min", and other operations remain unchanged.

[0081] Example 8

[0082] The present example provides a preparation method of Psidium fructus extract, which is only different from example 1 in that step (2) is "adding a compound enzyme (the compound enzyme includes neutral protease, xylanase, pectinase, tannase in a mass ratio of 2:2:1:1) to the product obtained in step (1), the addition amount of the compound enzyme is 2.5% of the mass of Psidium fructus, the enzymolysis temperature is 45 ℃, the enzymolysis time is 35 min, then keeping at 125 ℃, 0.15 MPa for 5 min", and other operations remain unchanged.

[0083] Example 9

[0084] The present example provides a preparation method of Psidium fructus extract, which is only different from example 1 in that step (3) is "passing the product obtained in step (2) through a 400-mesh screen, a 1-μm filter bag, and a 140-nm ceramic membrane, vacuum reducing pressure concentration, UHT ultra-high temperature instantaneous sterilization, freeze-drying, crushing and sieving, and then obtaining the product", and other operations remain unchanged.

[0085] Example 10

[0086] The present example provides a preparation method of Psidium fructus extract, which is only different from example 1 in that step (3) is "after the temperature drops to 35 ℃, passing the product obtained in step (2) through a 400-mesh screen, a 1-μm filter bag, and a 400-nm ceramic membrane, vacuum reducing pressure concentration, UHT ultra-high temperature instantaneous sterilization, freeze-drying, crushing and sieving, and then obtaining the product", and other operations remain unchanged.

[0087] Example 11

[0088] The present example provides a preparation method of Psidium fructus extract, which is only different from example 1 in that step (3) is "after the temperature drops to 35 ℃, passing the product obtained in step (2) through a 400-mesh screen, a 1-μm filter bag, and a 50-nm ceramic membrane, vacuum reducing pressure concentration, UHT ultra-high temperature instantaneous sterilization, freeze-drying, crushing and sieving, and then obtaining the product", and other operations remain unchanged.

[0089] Comparative Example 1

[0090] The comparative example provides a preparation method of Psidium fructus extract, which is only different from Example 1 in that step (1) is "crushing Psidium fructus, passing through a 200-mesh screen, ultrasonic extraction (ultrasonic power 400 W, time 40 min, temperature 50°C, solid-liquid ratio 1:20)", and other operations remain unchanged.

[0091] Comparative Example 2

[0092] The comparative example provides a preparation method of Psidium fructus extract, which comprises:

[0093] (1) crushing Psidium fructus, passing through a 200-mesh screen. Mixing Psidium fructus powder with water at a solid-liquid ratio of 1:20, heating and refluxing for extraction twice, each time for 40 min, to obtain an extraction liquid;

[0094] (2) passing the obtained product through a 400-mesh screen, a 1-μm filter bag, and a 140-nm ceramic membrane to obtain a filtrate;

[0095] (3) vacuum reducing pressure and concentrating the filtrate for sterilization, and then freeze-drying to obtain the Psidium fructus extract.

[0096] Comparative Example 3

[0097] The comparative example provides a preparation method of Psidium fructus extract, which comprises:

[0098] (1) crushing Psidium fructus, passing through a 200-mesh screen. Mixing Psidium fructus powder with 80% ethanol at a solid-liquid ratio of 1:20, ultrasonic extraction twice, each time for 40 min, to obtain an extraction liquid;

[0099] (2) passing the obtained product through a 400-mesh screen, a 1-μm filter bag, and a 140-nm ceramic membrane to obtain a filtrate;

[0100] (3) vacuum reducing pressure and concentrating the filtrate for sterilization, and then freeze-drying to obtain the Psidium fructus extract.

[0101] Comparative Example 4

[0102] The comparative example provides a preparation method of Psidium fructus extract, which is only different from Example 1 in that it does not contain step (2), and other operations remain unchanged.

[0103] Comparative Example 5

[0104] The comparative example provides a preparation method of Psidium fructus extract, which is only different from Example 1 in that step (1) is "crushing Psidium fructus, passing through a 200-mesh screen", and other operations remain unchanged.

[0105] Test Example 1

[0106] The products prepared in Examples 1-11 and Comparative Examples 1-5 were subjected to yield calculation and determination of polyphenol content, polysaccharide content, and turbidity value in this test example.

[0107] 1.1 Polyphenol content detection: Refer to national standard GB / T 8313. Take 1.0 mL of gallic acid working solution, water (as blank control), and test solution, add 5 mL of 10% Folin phenol reagent, react for 5 min, add 4 mL of 7.5% sodium carbonate solution to each, after constant volume, place at room temperature for 60 min, measure the absorbance at 765 nm wavelength, the average of three determinations was taken, and the polyphenol content was calculated. The results are shown in Table 1.

[0108] 1.2 Polysaccharide content detection method: Refer to national standard GB / T 40632. Accurately weigh 2.0 g of the sample of Examples 1-11 and Comparative Examples 1-5, dissolve in 50 mL of distilled water, add 95% ethanol in an amount of 4 times the volume of the dissolved solution, stand for 12 h, centrifuge at 2500 r / min for 15 min. After the precipitate is taken out after vacuum drying to constant weight in an 85 ℃ constant temperature oven, make up to 100 mL for standby. Accurately take 1 mL of glucose standard working solution, water, and sample to be tested in a 20 mL test tube, add 2.0 mL of phenol solution to the test solution, shake well, stand, then add 7.0 mL of concentrated sulfuric acid, mix well with a vortex mixer, heat in boiling water for 10 min, cool to room temperature in an ice water bath, stand for 30 min, measure the absorbance at 490 nm wavelength, detect three groups of parallel samples, take the average value, and calculate the polysaccharide content. The results are shown in Table 1.

[0109] 1.3 Turbidity value detection method: Accurately weigh 0.5 g of the sample of Examples 1-11 and Comparative Examples 1-5, add 100 mL of water, and prepare a 0.5% concentration sample solution for standby. A portable microcomputer turbidity meter (model: WGZ-1BW) was used for determination, and the turbidity value (NTU value) was read. The average of three determinations was taken, and the NTU value was calculated. The higher the NTU value, the higher the solution turbidity and the lower the clarity. The results are shown in Table 1.

[0110] Table 1

[0111]

[0112] From the data in Table 1, it can be seen that the extraction method of the present application has a high extraction rate for polyphenols and polysaccharides, and the yield is also high, and the product is clear. Microfluidization extraction and enzymolysis are indispensable, and the selection of enzymes is very important for the content of polyphenols and polysaccharides. Neutral protease, xylanase, pectinase and tannase have a mutually complementary effect and are indispensable. The temperature reduction before ceramic membrane filtration is very important for improving the content of polyphenols and clarity. The pore size of the ceramic membrane is also very important for the content of polyphenols and polysaccharides.

[0113] Test Example 2

[0114] Antioxidant effect test

[0115] The DPPH solution was dissolved in anhydrous ethanol to a concentration of 0.1 mg / mL, and the Psidium guajava young fruit extract solutions prepared in Examples 1-11 and Comparative Examples 1-5 (concentration: 1-0.03125 mg / mL) were prepared at the same time. 150 μL of the DPPH ethanol solution and 150 μL of the Psidium guajava young fruit extract solution were mixed uniformly, a blank control group was set, and the reaction was carried out at room temperature for 30 min in the dark, and then the mixture was shaken uniformly, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate of the sample was calculated according to the following formula:

[0116]

[0117] The concentration of the sample to be tested was taken as the X axis, and the absorbance was taken as the Y axis to prepare a scatter plot with a smooth line and data markers. The upper, middle and lower three points with a clearance rate of about 50% were selected to draw a straight line, and the straight line equation was obtained to calculate the DPPH free radical scavenging IC50 value and clearance rate of the sample. The DPPH free radical scavenging IC50 value results are shown in Table 2.

[0118] Table 2

[0119]

[0120] Test Example 3

[0121] Anti-aging effect test

[0122] A Psidium guajava young fruit extract solution with a concentration of 1 mg / ml was prepared. In a 96-well plate, 0.2 mg / mL of collagenase solution was added to 140 μL, and 60 μL of sample solution was added, and a 100% enzyme activity control group was set up, i.e. 60 μL of PBS was used instead of the test sample solution, and the same volume of buffer salt was used instead of the blank control and sample blank groups. After incubation at 37°C for 20 min, 40 μL of substrate FALGPA buffer salt solution was added, and the absorbance change at 330 nm for 0-20 min was measured. The collagenase inhibition rate was calculated according to the following formula:

[0123]

[0124] ΔA330B = difference between absorbance values of two time points (0 min and 20 mins) of control group.

[0125] ΔA330A = difference between absorbance values of two time points (0 min and 20 mins) of blank group.

[0126] ΔA330D = difference between absorbance values of two time points (0 min and 20 mins) of sample group.

[0127] ΔA330C = difference between absorbance values of two time points (0 min and 20 mins) of sample blank group.

[0128] The results were measured 3 times on average for each group, the average value was taken, and the collagenase inhibition rate value was calculated. The results are shown in Table 3.

[0129] Table 3

[0130]

[0131] Test Example 4

[0132] Anti-inflammatory effect test

[0133] A hyaluronidase solution of 0.25-1 mg / mL and a hyaluronic acid solution of 1 mg / mL were prepared by using 0.1 mM acetic acid buffer. A guava immature fruit extract solution prepared by using 1 mg / mL of Examples 1-11 and Comparative Examples 1-5 was prepared by using water as a solvent. The operation is shown in Table 4:

[0134] Table 4

[0135]

[0136]

[0137] A - absorbance value of sample solution

[0138] B - absorbance value of sample blank

[0139] C - absorbance value of control solution

[0140] D - absorbance value of control blank

[0141] Table 5

[0142]

[0143] The results were measured 3 times on average for each group, the average value was taken, and the collagenase inhibition rate value was calculated. The results are shown in Table 5.

[0144] Test Example 5

[0145] Anti-glycation test

[0146] A solution of the Psidium fructus extract prepared in Example 1-11 and Comparative Example 1-5 was prepared at 1 mg / mL in a phosphate buffer (50 mmol / L, pH 7.4). In a test tube, 1 mL of the sample to be tested, 0.8 mg / mL of a bovine serum albumin solution, 200 mM of a glucose solution, and the phosphate buffer were added, respectively, and heated at 60°C for 24 h, with aminoguanidine hydrochloride (AG) as a positive control. The fluorescent AGEs were measured using a fluorescence microplate reader, with an excitation wavelength of 370 nm and an emission wavelength of 420 nm. The content of the fluorescent AGEs was expressed as the fluorescence intensity AU, and the inhibition rate of the sample on the formation of the fluorescent AGEs was calculated using Formula (1):

[0147]

[0148] A0 — initial fluorescence intensity of the blank control group

[0149] A1 — initial fluorescence intensity of the experimental group

[0150] B0 — fluorescence intensity after reaction of the blank control group

[0151] B1 — fluorescence intensity after reaction of the experimental group

[0152] Table 6

[0153]

[0154] Test Example 6

[0155] HaCaT cell ROS scavenging effect determination

[0156] According to the CCK-8 determination results, the test concentration of the Psidium fructus extract obtained from Example 1 and Comparative Example 2 was selected as 125 µg / mL, and the intracellular reactive oxygen species ROS level of HaCaT cells was determined. Healthy cells in the exponential growth phase were selected, trypsinized and counted, and then inoculated into a 12-well plate at about 2.5×10 5 cells per well, and cultured for 24 hours until the cell growth reached 50%-70% coverage. Subsequently, the model group and the sample group were added with complete culture medium containing or not containing the test substance and PBS, respectively, and subjected to 2 h of UVA irradiation treatment (3T3 NRU phototoxic ultraviolet radiation meter, 350-400 nm wavelength, 2 mW / cm 2). After irradiation, all groups were placed in the incubator for 1 h. In addition, DCFH-DA was diluted to 10 micromole / liter using serum-free culture solution, and after incubating the cells for 20 minutes, they were treated with trypsin, centrifuged to discard the supernatant, and then 1 mL of PBS was added and centrifuged to collect, and finally 200 μL of PBS was added to each tube in the flow tube. The BD-FACSAriaTM Fusion flow cytometer was used for ROS detection.

[0157] The detection results are shown in Table 1. Figure 2 Compared with the blank control group, the ROS content of the model control group was significantly increased (p < 0.001), indicating that the model was successfully constructed. Compared with the model control group, the guava young fruit extract of Example 1 group (p < 0.001) and the guava young fruit extract of Comparative Example 2 group (p < 0.01) at a concentration of 125 μg / mL can significantly inhibit the generation of ROS, and have antioxidant effect; and the antioxidant effect of the guava young fruit extract of Example 1 group is better than that of Comparative Example 2 group.

[0158] Test Example 7

[0159] Anti-inflammatory efficacy determination of macrophage RAW264.7

[0160] According to the CCK-8 determination results, the test concentration of the guava young fruit extract obtained from Example 1 and Comparative Example 2 was selected as 125 μg / mL, and the anti-inflammatory efficacy determination of macrophage RAW264.7 was carried out. The ELISA method was used to detect the inhibitory effect of the sample on TNF-α, IL-6 and NO. The complete culture medium was used to configure the RAW264.7 cell suspension to 5×10 4 6 / mL, and it was inoculated in a 24-well plate at 1000 μL per well, and incubated for 24 hours. The blank control group, the positive control group (5 μM dexamethasone sodium acetate) and the sample group were set, and the latter two groups were added with the test substance according to the corresponding concentration of CV90 cell survival rate. After incubation in a 37 °C, 5% CO2 incubator for 1 hour, 1 μg / mL of lipopolysaccharide (LPS) was added to each well with a final concentration, and incubation was continued for 24 hours. After the end, the cell supernatant was collected by low-temperature centrifugation, and the contents of TNF-α, IL-6 and NO were detected using the ELISA kit and the nitric oxide detection kit according to the instruction steps.

[0161] The detection results are shown in Table 1. Figures 3-5The relative expression levels of NO, IL-6 and TNF-a in the model control group were significantly higher than those in the blank control group (p < 0.001), and the positive control group significantly inhibited the generation of NO and inflammatory factors in macrophages (p < 0.001) compared with the model control group, indicating that the model was successfully constructed. Compared with the model control group, the guava young fruit extract in Example 1 group and Comparative Example 2 group at a concentration of 125 μg / mL could significantly inhibit the generation of NO (p < 0.001), and could also inhibit the gene expression of inflammatory factors IL-6 (p < 0.001) and TNF-a (p < 0.001), and had significant anti-inflammatory efficacy. The anti-inflammatory effect of the guava young fruit extract in Example 1 group was significantly better than that in Comparative Example 2 group.

[0162] Test Example 8

[0163] Single Material Zebrafish Maximum Tolerated Concentration (MTC) Determination

[0164] Randomly selected 4 dpf wild type AB strain zebrafish were allocated to 6-well plates, and 30 zebrafish were treated in each well (experimental group). The samples were given in water (concentration see Table 7), and a normal control group was set up, with a volume of 3 mL per well. After 24 h of treatment at 28°C, the number of dead zebrafish in each experimental group was counted, and the MTC of the normal zebrafish was determined.

[0165] Table 7

[0166]

[0167] As can be seen from Table 7, the MTC of the guava young fruit extract prepared in Example 1 and Comparative Example 2 was 31.2 μg / mL.

[0168] Zebrafish Collagen Level Determination

[0169] Randomly selected 4 dpf wild type AB strain zebrafish were allocated to 6-well plates, and 30 zebrafish were treated in each well (experimental group). The samples of Example 1 and Comparative Example 2 were given in water (test concentration was 31.2 μg / mL), and a normal control group was set up, with a volume of 3 mL per well, and three parallel experiments were set up. After 24 h of treatment at 28°C, the zebrafish samples were collected according to the instructions of the collagen COL I ELISA kit, the data were collected by a multifunctional enzyme label instrument, and the collagen content in the zebrafish was analyzed.

[0170] The results are shown in Table 8. Figure 6As shown, compared with the normal control group, the collagen content of Example 1 group (p < 0.001) and Comparative Example 2 group (p < 0.01) was significantly increased. The collagen content of guava fruit extract in Example 1 increased by 88.94% at a concentration of 31.2 µg / mL, while the collagen content of guava fruit extract in Comparative Example 2 increased by 36.54% at the same concentration, indicating that the effect of Example 1 group was significantly better than that of Comparative Example 2 group.

[0171] Test Example 9

[0172] Zebrafish Inflammation Test

[0173] Three-day-first-flush (dpf) transgenic neutrophils were randomly selected and treated with green fluorescent MPX in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (test concentration 31.2 µg / mL) and a positive control (dexamethasone acetate 50.0 μM) were administered in aqueous solution. A normal control group and a model control group were also included. Each well contained 3 mL of fluid. Except for the normal control group, all experimental groups were treated with sodium dodecyl sulfate in aqueous solution to establish a zebrafish skin inflammation model. After treatment at 28 ℃ for 18 h, 10 zebrafish from each group were randomly selected and photographed under a fluorescence microscope. Images were then saved. Figure 8 Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils in zebrafish skin.

[0174] Depend on Figure 7 As shown, compared with the normal control group, the number of neutrophils in the zebrafish skin of the model control group was significantly increased (p < 0.001), and compared with the model control group, the dexamethasone acetate positive group significantly inhibited the aggregation of neutrophils in the skin, indicating that the model was successfully constructed. Compared with the model control group, the guava fruit extract of Example 1 group (p < 0.001) and Comparative Example 2 group (p < 0.01) significantly inhibited the aggregation of neutrophils at a concentration of 31.2 µg / mL, exhibiting anti-inflammatory effects; and the guava fruit extract of Example 1 group was superior to that of Comparative Example 2 group.

[0175] This extract is high in polyphenols and polysaccharides, and experimental verification has shown that it has significant antioxidant, anti-inflammatory, anti-aging, and anti-glycation effects. This extract can be used to prepare various formulations, such as beauty nutrients or skincare products, to improve skin inflammation and prevent skin aging. The extract powder of this invention has good flowability, high clarity after dissolving in water, stable properties, and no visible sediment or flocculent matter, making it suitable for various formulation types. Applicable formulation forms include: powders, granules, hard capsules, soft capsules, tablets, gummies, oral solutions, jellies, emulsions, beverages, masks, creams, serums, lotions, toners, and cosmetics.

[0176] The applicant declares that the present application is illustrated by the above-mentioned embodiments of the preparation method of Psidium guajava young fruit extract, its product and application, but the present application is not limited to the above-mentioned embodiments, i.e. it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0177] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0178] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable mode without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination modes.

Claims

1. A method of preparing a Psidium guajava young fruit extract, characterized by, The preparation method comprises: (1) crushing Psidium guajava young fruits, mixing with water, and then performing micro-jet extraction to obtain a first mixture; (2) mixing the first mixture with a composite enzyme, performing enzymolysis, and then performing incubation to obtain a second mixture; (3) performing ceramic membrane filtration on the second mixture, performing concentration under reduced pressure, sterilization, and drying, and then obtaining the Psidium guajava young fruit extract; the composite enzyme is a combination of neutral protease, xylanase, pectinase and tannase with a mass ratio of (2-3):(2-3):(1-2):(1-2); the pore size of the ceramic membrane is 100-220 nm, and the temperature during the ceramic membrane filtration of the second mixture is 30-40 ℃; the pressure during the incubation is 0.1-0.2 MPa, the temperature is 120-130 ℃, and the time is 10-20 min; the temperature of the micro-jet extraction is 30-60 ℃, and the number of times is 1-6 times; the pressure of the micro-jet extraction is 100-250 MPa, and the mass ratio of the material to the liquid is 1:(10-40); the mass of the composite enzyme is 2-3% of the mass of the Psidium guajava young fruits, the temperature of the enzymolysis is 40-50 ℃, and the time is 30-40 min. 2.A Psidium guajava young fruit extract prepared by the preparation method of the Psidium guajava young fruit extract according to claim 1. 3.Use of the Psidium guajava young fruit extract according to claim 2 in the preparation of a product with anti-aging, anti-inflammatory and anti-glycation effects, wherein the product is a cosmetic. 4.Use of the Psidium guajava young fruit extract according to claim 2 in the preparation of a health-care product with antioxidant effects.

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