A composition with anti-sugar and anti-aging efficacy and application thereof

Abstract

This invention relates to a composition with anti-glycation and anti-aging effects and its application. The composition comprises, by weight, 10-30 parts of fermented sacha inchi protein peptides, 20-60 parts of guava extract, and 5-20 parts of disodium pyrroloquinoline quinone. The fermented sacha inchi protein peptides are prepared by a method comprising the following steps: sacha inchi pulp is mixed with an organic solvent and defatted under subcritical conditions to obtain defatted sacha inchi; the defatted sacha inchi is mixed with water and pretreated using a microfluidic jet to obtain pretreated sacha inchi; the pretreated sacha inchi is subjected to ultrasonic extraction and water bath extraction sequentially, and the supernatant is collected by centrifugation to obtain sacha inchi protein; the sacha inchi protein is mixed with Bacillus subtilis for fermentation to obtain a fermentation broth; the fermentation broth is subjected to ultrasonic extraction, filtered, and dried to obtain the final product. The composition prepared by this invention has antioxidant, anti-glycation, and anti-aging effects, providing a functional ingredient source for the skincare industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a composition with anti-glycation and anti-aging effects and its application. Background Technology

[0002] Sacha indica ( Plukenetia volubilis Sacha inchi (L.), also known as South American oil vine, star oil vine, or Inca fruit, is a perennial woody vine belonging to the Euphorbiaceae family, native to the tropical rainforests of the Andes Mountains in South America. In 2006, Sacha inchi was introduced to Yunnan Province, my country from Peru, and large-scale trial planting has gradually established a certain planting area in suitable climatic regions such as Pu'er and Xishuangbanna. Studies have shown that Sacha inchi seeds are rich in oil and protein, with a high content of ω-3 unsaturated fatty acids in the oil. It also contains vitamin E, phytosterols, phenolic compounds, carotenoids, and other components with antioxidant, lipid-regulating, cardiovascular disease-preventing, immunity-enhancing, anti-inflammatory, and anti-tumor activities, making it a promising new natural health resource. However, current domestic development and utilization of Sacha inchi mainly focuses on primary products, primarily cold-pressed edible oil and roasted nuts, resulting in a limited product range and low added value. Although Yunnan has established a certain scale of Sacha inoculus cultivation, it has not yet formed a complete deep processing system and industrial chain. The high-quality protein resources in Sacha seeds are underutilized, and the systematic development and high-value utilization of its proteins and functional peptides are significantly lagging behind. There are few reports on Sacha peptide products in existing technologies, and a lack of mature, stable, and scalable preparation processes and standardized quality control systems limits the application of Sacha in high-value-added fields such as nutritional supplements and special diets. With the rapid growth in consumer demand for natural active ingredients such as plant proteins and functional peptides, developing highly active, delicious, safe Sacha peptide products derived from Yunnan's local Sacha resources with clear health benefits is of great significance for improving the economic benefits of Sacha cultivation, extending the industrial chain of Yunnan's characteristic plants, and promoting the development of the regional health industry. Therefore, it is necessary to address the problems of insufficient utilization and narrow application of Sacha protein resources by providing a high-value-added Sacha peptide product and its preparation process to achieve efficient, precise, and diversified development of Sacha resources.

[0003] Under modern high-sugar diets, excess free sugars can undergo non-enzymatic glycation reactions with proteins, lipids, or nucleic acids without enzymatic catalysis, generating advanced glycation end products (AGEs). AGEs, as stubborn "glycation toxins," not only accelerate the functional degeneration of multiple tissues such as the skin, cardiovascular system, nervous system, and immune system, but also contribute to various problems. For example, in the skin, they cross-link with collagen and elastin, activating matrix metalloproteinases (MMPs) and leading to sagging and wrinkles; in blood vessels, they harden blood vessel walls, increasing the risk of atherosclerosis; in the brain, they interfere with neurotransmitter transmission, promoting cognitive decline and neurodegenerative diseases such as Alzheimer's disease; and in the immune system, they weaken the ability of white blood cells to clear pathogens and induce chronic inflammation. Furthermore, they create a vicious cycle through inflammation and oxidative stress, accelerating systemic aging. Although various synthetic anti-glycation agents such as aminoguanidine, metformin, and pioglitazone have been developed, their clinical translation remains limited. In contrast, natural active ingredients with wide availability, low toxicity, and multi-target synergistic effects have become the focus of anti-glycation drug development: polyphenols can scavenge active carbonyl groups and free radicals by relying on ortho-hydroxyl groups and conjugated double bonds; polysaccharides can form stable complexes with glycosylation intermediates by utilizing functional groups such as hydroxyl and carboxyl groups; and peptides can selectively capture key intermediates such as glyoxal and methylglyoxal through specific amino acid residues, thereby blocking the AGEs generation pathway and providing a safe and promising strategy for the prevention and control of chronic metabolic diseases and aging related to glycation.

[0004] CN115869205B discloses an anti-non-enzymatic glycosylation composition and its application, as well as a non-enzymatic glycosylation inhibitor. The anti-non-enzymatic glycosylation composition, by weight, comprises the following raw materials: 30-90 parts sialic acid, 10-40 parts ginkgo biloba extract, 10-40 parts acerola cherry extract, and 0.1-10 parts rosemary extract. Through specific proportions, the composition can achieve an anti-non-enzymatic glycosylation level comparable to existing technologies, while also improving stability. However, sialic acid, as the main component, may have limited availability or high cost.

[0005] CN113332354B discloses an anti-glycation and anti-aging fermented composition and its preparation method. By weight, it comprises 40-120 parts of chamomile flower, 40-120 parts of houttuynia cordata, 20-100 parts of amla, 10-80 parts of hawthorn, 0.1-3 parts of fermentation starter culture, 6-40 parts of enzyme preparation, and 1500-3000 parts of water. Through enzymatic hydrolysis and fermentation, the effective content of the active ingredients quercetin, chlorogenic acid, and gallic acid is increased, solving the problem of low utilization rate of active ingredients in existing products and significantly improving the anti-glycation and anti-aging effects.

[0006] CN101686954B discloses an inhibitory effect on AGEs formation, developing a composition containing plant extracts, including Epimedium, Rumex acetosa, Smilax china, and grape seed extract, to prevent or resist protein glycosylation. By inhibiting AGEs formation, it combines with substances such as aminoguanidine, EDTA derivatives, and inositol hexaphosphate to form a cosmetic or pharmaceutical composition that can be used topically or orally. This composition effectively inhibits protein glycosylation, slows down the aging process of tissues, improves skin elasticity and plasticity, and reduces albumin excretion in diabetic patients. While this patent introduces chemically synthesized components such as aminoguanidine and EDTA derivatives to enhance the AGEs inhibitory effect, long-term oral administration of aminoguanidine, a diabetes treatment drug, may cause liver and kidney toxicity (such as inducing anemia and neuropathy). Furthermore, EDTA, as a metal chelating agent, may interfere with the body's absorption of essential trace elements (such as calcium and iron), making it unsuitable for long-term use. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a composition with anti-glycation and anti-aging effects and its application.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a composition having anti-glycation and anti-aging effects, wherein the composition comprises, by weight parts, 10-30 parts of Sacha inca protein fermentation peptide, 20-60 parts of guava extract and 5-20 parts of disodium pyrroloquinoline quinone.

[0010] The sacha indica protein fermentation peptides were prepared by a method comprising the following steps:

[0011] (1) The sacha inophyllum pulp was mixed with an organic solvent and defatted under subcritical conditions to obtain defatted sacha inophyllum;

[0012] (2) Mix defatted Sacha in water and perform microjet pretreatment to obtain pretreated Sacha in water;

[0013] (3) The pretreated Sacha in a series of steps were subjected to ultrasonic extraction and water bath extraction, and the supernatant was collected by centrifugation to obtain Sacha in a series of steps.

[0014] (4) Mix Sacha inophylline protein with Bacillus subtilis and ferment to obtain fermentation broth;

[0015] (5) The fermentation broth is extracted by ultrasonication, filtered, and dried to obtain the final product.

[0016] This invention is the first to combine fermented povidone-iodine protein peptides, guava extract, and disodium pyrroloquinoline quinone (PQQ) to obtain a synergistic composition. This composition achieves multi-target synergy through rational compounding: on the one hand, it can synergistically capture key glycation intermediates such as acetone aldehyde and glyoxal and chelate iron ions, delaying the non-enzymatic glycation process and inhibiting the formation of advanced glycation end products (AGEs); on the other hand, it can scavenge reactive oxygen species, inhibit matrix metalloproteinases (MMPs) activity, and slow down collagen degradation and cross-linking, thereby exerting significant anti-glycation and anti-aging effects and exhibiting a synergistic effect. Furthermore, this composition has a simplified formulation but forms a complete closed-loop mechanism of action, with clearly defined functional indicators, good dosage form adaptability and industrialization feasibility, highlighting its scientific rigor and systematic nature, demonstrating strong technological and product innovation advantages, and providing a new solution for the development of anti-glycation and anti-aging health products based on natural active ingredients.

[0017] The mass fractions of fermented sacha inchi protein peptides can be selected from 10, 12, 15, 18, 20, 22, 25, 28, and 30 parts, etc. The mass fractions of guava extract can be selected from 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, and 60 parts, etc. The mass fractions of disodium pyrroloquinoline quinone can be selected from 5, 8, 10, 12, 15, 18, and 20 parts, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0018] By using microfluidic and bio-fermentation technologies, sacha inchi protein is directionally broken down into small molecule active peptides, which are more easily absorbed by the human intestine and significantly improve its bioavailability, antioxidant and anti-glycation activities.

[0019] Preferably, the subcritical temperature is 40-50 °C, the pressure is 0.4-0.5 MPa, and the time is 40-60 min.

[0020] Temperatures can be selected from 40 ℃, 42 ℃, 46 ℃, 48 ℃, 50 ℃, etc.; pressures can be selected from 0.4 MPa, 0.42 MPa, 0.44 MPa, 0.46 MPa, 0.48 MPa, 0.5 MPa, etc.; and time can be selected from 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc. Other specific values ​​within the above ranges can also be selected, which will not be elaborated here.

[0021] Preferably, the ratio of the sacha inophyllum pulp to the organic solvent is 1 g:(1-1.5) mL, and the organic solvent includes butane or propane.

[0022] The specific point values ​​in (1-1.5) can all be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0023] Preferably, the subcritical treatment is performed 4-5 times.

[0024] Preferably, the ratio of defatted Sacha in water to water is 1 g:(15-30) mL. The specific values ​​in (15-30) can be selected from 15, 18, 20, 22, 25, 28, 30, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0025] Preferably, the pressure of the microjet is 100-300 MPa, the temperature is 50-60 ℃, and the number of jets is 2-6.

[0026] The pressure can be selected from 100 MPa, 120 MPa, 150 MPa, 180 MPa, 200 MPa, 220 MPa, 250 MPa, 280 MPa, 300 MPa, etc., the temperature can be selected from 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, etc., and the number of cycles can be selected from 2, 3, 4, 5, 6, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0027] Preferably, step (3) before ultrasonic extraction further includes adjusting the pH to 9-11, such as 9, 9.2, 9.5, 9.8, 10, 10.2, 10.5, 10.8, 11, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0028] Preferably, the temperature of the ultrasound in step (3) is 40-60 ℃, the time is 30-60 min, and the power is 400-800W.

[0029] The ultrasound temperature can be selected from 40 ℃, 42 ℃, 45 ℃, 48 ℃, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃, etc., the time can be selected from 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc., and the power can be selected from 400 W, 480 W, 560 W, 640 W, 720 W, 800 W, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0030] Preferably, the water bath extraction in step (3) is performed at a temperature of 45-55 °C for 30-60 min.

[0031] Temperatures can be selected from 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, etc., and time can be selected from 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0032] Preferably, after the water bath extraction in step (3), the precipitate obtained by centrifugation is further mixed with water at a ratio of 1 g:(10-15) mL, the pH is adjusted to 9-11, ultrasonic extraction is performed at 40-60 ℃ for 30-60 min with an ultrasonic power of 400-800 W, water bath extraction is performed at 45-55 ℃ for 30-60 min, and the supernatant is collected by centrifugation.

[0033] The specific point values ​​in (10-15) can be selected as 10, 11, 12, 13, 14, 15, etc.; the pH can be selected as 9, 9.2, 9.5, 9.8, 10, 10.2, 10.5, 10.8, 11, etc.; the ultrasonic temperature can be selected as 40 ℃, 42 ℃, 44 ℃, 46 ℃, 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, etc.; the time can be selected as 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc.; the power can be selected as 400W, 480 W, 560 W, 640 W, 720 W, 800 W, etc.; and the water bath extraction temperature can be selected as 45 ℃, 48 ℃, 50 ℃, 52 ℃, 55 ℃, etc. Temperature, etc., and time can be selected as 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0034] Preferably, step (3) further includes adjusting the pH to 4-4.6 before sedimentation, such as 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0035] Preferably, the settling temperature in step (3) is 4-8 ℃ and the time is 16-32 h.

[0036] Temperatures can be selected from 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, etc., and time can be selected from 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0037] Preferably, step (4) further includes mixing sacha inoculin with water at a ratio of 1 g:(15-25) mL, dialysis to remove salt, sterilization, and obtaining a sacha inoculin aqueous solution.

[0038] The specific point values ​​in (15-25) can all be selected from 15, 18, 20, 22, 25, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0039] Preferably, the pH of the sacha inophylline protein aqueous solution is 7-7.5, such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0040] Preferably, the dialysis bag used for dialysis has a molecular weight of 500-1000 Da, and the dialysis time is 24-48 h.

[0041] The molecular weight can be selected from 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, etc., and the time can be selected from 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0042] Preferably, the fermentation temperature is 25-37 ℃ and the time is 18-48 h.

[0043] Temperatures can be selected from 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, etc., and time can be selected from 18 h, 20 h, 22 h, 25 h, 28 h, 30 h, 32 h, 35 h, 38 h, 40 h, 42 h, 45 h, 48 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0044] Preferably, the volume percentage of Bacillus subtilis culture in the fermentation system is 0.5-3%, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0045] Preferably, the viable count of the Bacillus subtilis bacterial solution is 1×10⁻⁶. 7 -5×10 7 CFU / mL, for example 1×10 7 CFU / mL, 1.5×10 7 CFU / mL, 2×10 7 CFU / mL, 2.5×10 7 CFU / mL, 3×10 7 CFU / mL, 3.5×10 7 CFU / mL, 4×10 7 CFU / mL, 4.5×10 7 CFU / mL, 5×10 7 CFU / mL, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0046] Preferably, the temperature of the ultrasound in step (5) is 40-50 ℃, the time is 30-45 min, and the power is 400-800W.

[0047] Temperatures can be selected from 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, etc.; time can be selected from 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, etc.; power can be selected from 400 W, 480 W, 560 W, 640 W, 720 W, 800 W, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0048] Preferably, the guava extract is prepared by a method comprising the following steps:

[0049] (1) Guava was pretreated by steam explosion to obtain pretreated guava;

[0050] (2) Mix the pretreated guava with water, heat and reflux to extract, filter, concentrate and dry to obtain the product.

[0051] Guava extract obtained through steam explosion treatment can significantly increase its content of active ingredients and anti-aging activity.

[0052] Preferably, the water content of the guava in step (1) is 15-30 wt%, such as 15 wt%, 18 wt%, 20 wt%, 22 wt%, 25 wt%, 28 wt%, 30 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0053] Preferably, the steam explosion is performed at a pressure of 0.2-0.5 MPa, a temperature of 130-160 ℃, and a time of 0.5-2 min.

[0054] The pressure can be selected from 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, etc., the temperature can be selected from 130 ℃, 132 ℃, 135 ℃, 138 ℃, 140 ℃, 142 ℃, 145 ℃, 148 ℃, 150 ℃, 152 ℃, 155 ℃, 158 ℃, 160 ℃, etc., and the time can be selected from 0.5 min, 0.8 min, 1 min, 1.2 min, 1.5 min, 1.8 min, 2 min, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0055] Preferably, the process after steam explosion further includes drying.

[0056] Preferably, the water content of the pretreated guava is 5-10 wt%, such as 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0057] Preferably, the ratio of pretreated guava to water is 1 g:(15-30) mL, and the reflux extraction time is 1-2 h.

[0058] The specific point values ​​in (15-30) can be selected from 15, 18, 20, 22, 25, 28, 30, etc., and the time can be selected from 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0059] In a second aspect, the present invention provides the use of the composition with anti-glycation and anti-aging effects according to the first aspect in the preparation of cosmetic products with anti-glycation, anti-oxidation or anti-aging effects.

[0060] Preferably, the beauty product includes cosmetics or skin care products.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] (1) The fermented peptides of Sacha incisum protein provided by the present invention adopt the process route of ultrasonic extraction combined with Bacillus subtilis fermentation. The process conditions are mild, green and controllable, which can reduce the problems of bitter peptides, poor product stability and low bioavailability in traditional enzymatic hydrolysis, and significantly improve the antioxidant, anti-aging and anti-glycation activities of the product.

[0063] (2) The fermented protein peptides of Sacha involucrata obtained in this invention can effectively improve the DPPH free radical scavenging ability and collagenase inhibition ability in an in vitro model, and significantly reduce the generation of AGEs. At the same time, this invention realizes the high-value utilization of Sacha involucrata seeds, expanding them from a single primary agricultural product or oil raw material to a high-value-added active peptide functional raw material, enriching its deep processing application path in cosmetics and other fields, and helping to improve the overall economic benefits of the industry.

[0064] (3) This invention is the first to apply steam explosion technology to the extraction of active ingredients from guava. Through physical modification, the cell walls of the raw materials are efficiently destroyed, accelerating the release of active ingredients. Compared with traditional methods, this significantly improves the extraction yield and production efficiency of the target active ingredients. Moreover, the process uses water as the only extraction solvent, avoiding the risk of organic solvent residue. The obtained guava extract shows significant advantages in DPPH free radical scavenging, collagenase activity inhibition, and AGEs inhibition.

[0065] (4) This invention is the first to combine three components: Sacha inchi protein fermentation peptide, guava extract, and PQQ. The resulting composition can synergistically capture key glycosylation intermediates such as MGO and GO, chelate iron ions, and scavenge DPPH free radicals, achieving an AGEs inhibition rate of over 85%. Simultaneously, it can inhibit collagenase activity and regulate the expression levels of intracellular MMP-1, MMP-3, and MMP-9. This composition, through multi-target and multi-pathway synergistic effects, precisely intervenes in the glycosylation-oxidation-related aging network, achieving significant synergistic effects in antioxidation, inhibition of non-enzymatic glycosylation, and delaying aging.

[0066] (5) The composition of the present invention has the characteristics of simple composition, closed-loop mechanism, clear activity and strong dosage form adaptability. The active solution based on natural active ingredients is in line with the trend of the big health industry and has broad prospects for industrial application. Attached Figure Description

[0067] Figure 1 This is the result of the iron ion chelating ability test. *** P <0.001 indicates a significant difference compared to Application Example 1.

[0068] Figure 2 These are the results of MGO and GO capture capability tests. ** P <0.01、 *** P <0.001 indicates a significant difference compared to Application Example 1.

[0069] Figure 3 This is the result of the composition's anti-aging effect. # P <0.05、 ### P <0.001 indicates a significant difference compared to the control group; * P <0.05、 ** P <0.01、 *** P <0.001 indicates a significant difference compared to the model group; ++ P <0.01、 +++ P <0.001 indicates a significant difference compared to Application Example 1. Figure (A) shows the relative expression level of MMP1 mRNA, Figure (B) shows the relative expression level of MMP3 mRNA, and Figure (C) shows the relative expression level of MMP9 mRNA. Detailed Implementation

[0070] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0071] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0072] Bacillus subtilis Bacillus subtilis CICC 20030, Bacillus subtilis Bacillus subtilis CICC 24713, Bacillus subtilis Bacillus subtilis CICC 21927 was purchased from the China Industrial Microbial Culture Collection Center.

[0073] Leuconostoc mesenteroides ( Leuconostoc mesenteroides Purchased from the China Industrial Microbial Culture Collection Center, accession number CICC 6055.

[0074] Example 1

[0075] This embodiment provides a method for preparing fermented peptides from Sacha indica protein, the preparation method comprising:

[0076] (1) The sacha indica fruit pulp was subjected to subcritical defatting with butane as the defatting solvent and a material-to-liquid ratio of 1 g:1.2 mL. It was extracted at 45℃ and 0.45 MPa for 50 min and then subjected to dynamic circulation countercurrent extraction 4 times to obtain defatted sacha indica fruit.

[0077] (2) Defatted Sacha incisa fruit was mixed with water at a ratio of 1 g: 18 mL and subjected to high-pressure microjet extraction at a pressure of 200 MPa and a temperature of 55 ℃ for 4 times to obtain pretreated Sacha incisa fruit.

[0078] (3) Adjust the pH of the pretreated Sacha indica fruit to 10 and perform ultrasonic extraction. The ultrasonic temperature is 40 ℃, the time is 60 min, the power is 560 W, the extraction is performed in a 50 ℃ water bath for 45 min, and the supernatant is collected after centrifugation at 5000 rpm for 10 min.

[0079] (4) Mix the precipitate obtained in step (3) with water at a ratio of 1g:10mL, adjust the pH to 10, and perform ultrasonic extraction. The ultrasonic temperature is 40℃, the time is 60 min, the power is 560 W, the extraction is performed in a 50℃ water bath for 45 min, and the supernatant is collected after centrifugation at 5000 rpm for 10 min.

[0080] (5) Combine the supernatants obtained in steps (3) and (4), adjust the pH to 4.2, precipitate the protein at 4 °C overnight, centrifuge at 5000 rpm for 10 min, wash the precipitate, adjust the pH to 7, reconstitute the protein, the ratio of protein to water is 1 g: 20 mL, dialyze to remove salt using a 1000 Da dialysis bag to obtain saponin aqueous solution;

[0081] (6) After sterilizing the aqueous solution of sacha inophylline obtained in step (5), it is mixed with Bacillus subtilis CICC 20030 bacterial solution for fermentation. The fermentation temperature is 30 ℃, the fermentation time is 30 h, the inoculum amount is 3%, and the viable count of the bacterial solution is 3×10⁻⁶. 7 CFU / mL was used to obtain the fermentation broth;

[0082] (7) The fermentation broth was subjected to ultrasonic extraction for 35 min at 45 ℃ and 640 W. After filtration through a 220 nm ceramic membrane, the broth was concentrated and dried under reduced pressure and sterilized to obtain the Sacha Inchi protein peptide.

[0083] Example 2

[0084] This embodiment provides a method for preparing fermented peptides from Sacha indica protein, the preparation method comprising:

[0085] (1) The sacha indica fruit pulp was subjected to subcritical defatting with butane as the defatting solvent and a material-to-liquid ratio of 1 g:1 mL. It was extracted at 40 °C and 0.4 MPa for 60 min and then subjected to dynamic circulation countercurrent extraction 4 times to obtain defatted sacha indica fruit.

[0086] (2) Defatted Sacha incisa fruit was mixed with water at a ratio of 1 g: 15 mL and subjected to high-pressure microjet extraction at a pressure of 100 MPa and a temperature of 50 ℃ for 6 times to obtain pretreated Sacha incisa fruit.

[0087] (3) Adjust the pH of the pretreated Sacha indica fruit to 9, and perform ultrasonic extraction. The ultrasonic temperature is 50 ℃, the time is 50 min, the power is 480 W, the extraction is performed in a 55 ℃ water bath for 30 min, and the supernatant is collected after centrifugation at 5000 rpm for 10 min.

[0088] (4) Mix the precipitate obtained in step (3) with water, with a material-to-liquid ratio of 1g:12mL, adjust the pH to 9, and perform ultrasonic extraction. The ultrasonic temperature is 50℃, the power is 480W, the time is 50min, the extraction is performed in a 55℃ water bath for 30min, and the supernatant is collected after centrifugation at 5000 rpm for 10min.

[0089] (5) Combine the supernatants obtained in steps (3) and (4), adjust the pH to 4.6, precipitate the protein at 4 °C overnight, centrifuge at 5000 rpm for 10 min, wash the precipitate, adjust the pH to 7.5, reconstitute the protein, the protein to water ratio is 1 g: 15 mL, dialyze to remove salt using a 1000 Da dialysis bag to obtain saponin aqueous solution;

[0090] (6) After sterilizing the aqueous solution of sacha inophylline obtained in step (5), it is mixed with Bacillus subtilis CICC 20030 bacterial culture for fermentation. The fermentation temperature is 25 ℃, the time is 48 h, the inoculum size is 3%, and the viable count of the bacterial culture is 1×10⁻⁶. 7 CFU / mL was used to obtain the fermentation broth;

[0091] (7) The fermentation broth was subjected to ultrasonic extraction for 45 min at 40 ℃ and 640 W. After filtration through a 220 nm ceramic membrane, the broth was concentrated and dried under reduced pressure and sterilized to obtain the Sacha Inchi protein peptide.

[0092] Example 3

[0093] This embodiment provides a method for preparing fermented peptides from Sacha indica protein, the preparation method comprising:

[0094] (1) The sacha indica fruit pulp was subjected to subcritical defatting with butane as the defatting solvent and a material-to-liquid ratio of 1 g:1.5 mL. It was extracted at 50 °C and 0.5 MPa for 40 min and then subjected to dynamic circulation countercurrent extraction 4 times to obtain defatted sacha indica fruit.

[0095] (2) Defatted Sacha incisa fruit was mixed with water at a ratio of 1 g: 30 mL and subjected to high-pressure microjet extraction at a pressure of 300 MPa and a temperature of 60 ℃ for 3 times to obtain pretreated Sacha incisa fruit.

[0096] (3) Adjust the pH of the pretreated Sacha indica fruit to 11 and perform ultrasonic extraction. The ultrasonic temperature is 60℃, the time is 30min, the power is 800W, the extraction is performed in a 45℃ water bath for 1 h, and the supernatant is collected after centrifugation at 5000 rpm for 10 min.

[0097] (4) Mix the precipitate obtained in step (3) with water, with a material-to-liquid ratio of 1 g: 15 mL, adjust the pH to 11, and perform ultrasonic extraction. The ultrasonic temperature is 60 °C, the time is 30 min, the power is 800 W, and the extraction is performed in a 45 °C water bath for 1 h. After centrifugation at 5000 rpm for 10 min, the supernatant is collected.

[0098] (5) Combine the supernatants obtained in steps (3) and (4), adjust the pH to 4, precipitate the protein overnight at 4°C, centrifuge at 5000 rpm for 10 min, wash the precipitate, adjust the pH to 7.2, reconstitute the protein, the protein to water ratio is 1 g: 25 mL, dialyze to remove salt using a 1000 Da dialysis bag to obtain saponin aqueous solution;

[0099] (6) After sterilizing the aqueous solution of sacha inophylline obtained in step (5), it is mixed with Bacillus subtilis CICC 20030 bacterial solution for fermentation. The fermentation temperature is 37 ℃, the time is 20 h, the inoculum amount is 3%, and the viable count of the bacterial solution is 5 × 10⁻⁶. 7 CFU / mL was used to obtain the fermentation broth;

[0100] (7) The fermentation broth was subjected to ultrasonic extraction for 30 min at 50 ℃ and 640 W. After filtration through a 220 nm ceramic membrane, the broth was concentrated and dried under reduced pressure and sterilized to obtain the Sacha Inchi protein peptide.

[0101] Example 4

[0102] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (1) is to "subcritically defatt the Sacha indica pulp, using butane as the defatting solvent, with a material-to-liquid ratio of 1 g:1.2 mL, extracting at 30 ℃ and 0.3 MPa for 50 min, and then dynamically circulating countercurrently extracting 4 times to obtain defatted Sacha indica pulp". Other operations remain unchanged.

[0103] Example 5

[0104] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (1) is to "subcritically defatt the Sacha indica pulp, using butane as the defatting solvent, with a material-to-liquid ratio of 1 g:1.2 mL, extracting at 60 ℃ and 0.6 MPa for 50 min, and then dynamically circulating countercurrently extracting 4 times to obtain defatted Sacha indica pulp". Other operations remain unchanged.

[0105] Example 6

[0106] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this embodiment and Example 1 is that step (2) is to "mix defatted Sacha indica with water at a ratio of 1 g:18 mL, perform high-pressure microjet extraction at a pressure of 50 MPa and a temperature of 45 °C for 4 times to obtain pretreated Sacha indica", while other operations remain unchanged.

[0107] Example 7

[0108] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (2) is to "mix defatted Sacha indica with water at a ratio of 1 g:18 mL, perform high-pressure microjet extraction at a pressure of 400 MPa and a temperature of 65 °C for 4 times to obtain pretreated Sacha indica", while other operations remain unchanged.

[0109] Example 8

[0110] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (5) is: "combine the supernatants obtained in steps (3) and (4), adjust the pH to 3.5, precipitate the protein overnight at 4 °C, centrifuge at 5000 rpm for 10 min, wash the precipitate, adjust the pH to 7, reconstitute the protein, the ratio of protein to water is 1 g: 20 mL, and use a 1000 Da dialysis bag for dialysis to remove salt, to obtain an aqueous solution of Sacha indica protein". Other operations remain unchanged.

[0111] Example 9

[0112] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (5) is: "combine the supernatants obtained in steps (3) and (4), adjust the pH to 5, precipitate the protein overnight at 4°C, centrifuge at 5000 rpm for 10 min, wash the precipitate, adjust the pH to 7, reconstitute the protein, the ratio of protein to water is 1 g: 20 mL, dialyze to remove salt using a 1000 Da dialysis bag, and obtain an aqueous solution of Sacha indica protein". Other operations remain unchanged.

[0113] Example 10

[0114] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (6) is: "After sterilizing the aqueous solution of Sacha indica protein obtained in step (5), it is mixed with Bacillus subtilis CICC 24713 bacterial solution for fermentation. The fermentation temperature is 30 ℃, the fermentation time is 30 h, the inoculum amount of the bacterial solution is 3%, and the viable count of the bacterial solution is 3×10⁻⁶". 7 "CFU / mL, to obtain fermentation broth", other operations remain unchanged.

[0115] Example 11

[0116] This embodiment provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (6) is: "After sterilizing the aqueous solution of Sacha indica protein obtained in step (5), it is mixed with Bacillus subtilis CICC 21927 bacterial solution for fermentation. The fermentation temperature is 30 ℃, the time is 30 h, the inoculum amount of the bacterial solution is 3%, and the viable count of the bacterial solution is 3×10⁻⁶". 7"CFU / mL, to obtain fermentation broth", other operations remain unchanged.

[0117] Comparative Example 1

[0118] This comparative example provides a method for preparing fermented peptides of Sacha indica protein. The only difference between this method and Example 1 is that step (6) is: "After sterilizing the aqueous solution of Sacha indica protein obtained in step (5), it is mixed with Leuconostoc mesenteroides CICC 6055 bacterial solution for fermentation. The fermentation temperature is 30 ℃, the time is 30 h, the inoculum amount of the bacterial solution is 3%, and the viable count of the bacterial solution is 3×10⁻⁶". 7 "CFU / mL, to obtain fermentation broth", other operations remain unchanged.

[0119] Comparative Example 2

[0120] This comparative example provides a method for preparing fermented peptides of Sacha indica protein, which differs from Example 1 only in that step (1) is omitted, while other operations remain unchanged.

[0121] Comparative Example 3

[0122] This comparative example provides a method for preparing fermented peptides of Sacha indica protein, which differs from Example 1 only in that step (2) is omitted, while other operations remain unchanged.

[0123] Comparative Example 4

[0124] This comparative example provides a method for preparing fermented peptides of Sacha indica protein, which differs from Example 1 only in that step (6) is omitted, while other operations remain unchanged.

[0125] Preparation Example 1

[0126] This preparation example provides a method for preparing guava extract, the method comprising:

[0127] (1) The moisture content of guava medicinal material was controlled at 20 wt%, and steam explosion treatment was carried out. The steam explosion pressure was 0.3 MPa, the temperature was 145 ℃, the time was 1 min, the pressure release rate was less than 0.1 s, and the explosion was repeated once. The material was dried until the moisture content was 6 wt%.

[0128] (2) Mix the steam-exploded guava with water at a ratio of 1 g: 20 mL, heat under reflux for 1.5 h, extract twice, cool and filter through 600 mesh and 5 μm filter bags, concentrate under vacuum, sterilize and dry to obtain guava extract.

[0129] Preparation Example 2

[0130] This preparation example provides a method for preparing guava extract, the method comprising:

[0131] (1) The moisture content of guava medicinal material was controlled at 15 wt%, and steam explosion treatment was carried out. The steam explosion pressure was 0.2 MPa, the temperature was 130 ℃, the time was 2 min, the pressure release rate was less than 0.1 s, and the explosion was repeated once. The material was dried until the moisture content was 5 wt%.

[0132] (2) Mix the steam-exploded guava with water at a ratio of 1 g: 15 mL, heat and reflux for 1 h, extract twice, cool and filter through 600 mesh and 5 μm filter bags, concentrate under vacuum, sterilize and dry to obtain guava extract.

[0133] Preparation Example 3

[0134] This preparation example provides a method for preparing guava extract, the method comprising:

[0135] (1) The moisture content of guava medicinal material was controlled at 30 wt%, and steam explosion treatment was carried out. The steam explosion pressure was 0.5 MPa, the temperature was 160℃, the time was 0.5 min, the pressure release rate was less than 0.1 s, and the explosion was repeated once. The material was dried until the moisture content was 10 wt%.

[0136] (2) Mix the steam-exploded guava with water at a ratio of 1 g: 30 mL, heat and reflux for 2 h, extract twice, cool and filter through 600 mesh and 5 μm filter bags, concentrate under vacuum, sterilize and dry to obtain guava extract.

[0137] Preparation Example 4

[0138] This preparation example provides a method for preparing guava extract, which differs from preparation example 1 only in that step (1) is to "control the moisture content of guava medicinal material to 10 wt%, perform steam explosion treatment, the steam explosion pressure is 0.3 MPa, the temperature is 145 ℃, the time is 1 min, the pressure release rate is less than 0.1 s, cyclic explosion is performed once, and dried to a moisture content of 6 wt%", while other operations remain unchanged.

[0139] Preparation Example 5

[0140] This preparation example provides a method for preparing guava extract, which differs from preparation example 1 only in that step (1) is to "control the moisture content of guava medicinal material to 40 wt%, perform steam explosion treatment, the steam explosion pressure is 0.3 MPa, the temperature is 145 ℃, the time is 1 min, the pressure release rate is less than 0.1 s, cyclic explosion is performed once, and dried to a moisture content of 6 wt%", while other operations remain unchanged.

[0141] Preparation Example 6

[0142] This preparation example provides a method for preparing guava extract, which differs from Preparation Example 1 only in that step (1) is to "control the moisture content of guava medicinal material to 20 wt%, perform steam explosion treatment, the steam explosion pressure is 0.1 MPa, the temperature is 120 ℃, the time is 1 min, the pressure release rate is less than 0.1 s, cyclic explosion is performed once, and the water content is dried to 6 wt%", while other operations remain unchanged.

[0143] Preparation Example 7

[0144] This preparation example provides a method for preparing guava extract, which differs from Preparation Example 1 only in that step (1) is to "control the moisture content of guava medicinal material to 20 wt%, perform steam explosion treatment, the steam explosion pressure is 0.7 MPa, the temperature is 170 ℃, the time is 1 min, the pressure release rate is less than 0.1 s, cyclic explosion is performed once, and the water content is dried to 6 wt%", while other operations remain unchanged.

[0145] Preparation Example 8

[0146] This preparation example provides a method for preparing guava extract, which differs from Preparation Example 1 only in that step (1) is to "control the moisture content of guava medicinal material to 20 wt%, perform high temperature and high pressure treatment, with a pressure of 0.3 MPa, a temperature of 145 ℃, a time of 1 min, and dry to a moisture content of 6 wt%", while other operations remain unchanged.

[0147] Preparation Example 9

[0148] This preparation example provides a method for preparing guava extract, which differs from Preparation Example 1 only in that step (1) is "drying guava medicinal material to a moisture content of 6 wt%", while other operations remain unchanged.

[0149] Preparation Example 10

[0150] This preparation example provides a method for preparing guava extract. The only difference between this method and Preparation Example 1 is that step (2) is to "mix the steam-exploded guava with water at a ratio of 1 g: 20 mL, perform ultrasonic extraction at a power of 720 W, a temperature of 60 °C, and a time of 45 min, extract twice, cool and filter through a 600-mesh and a 5 μm filter bag, concentrate under vacuum, sterilize and dry to obtain guava extract". Other operations remain unchanged.

[0151] Application Example 1

[0152] This application example provides a composition with anti-glycation and anti-aging effects, which includes, by mass parts, 20 parts of Sacha inca protein fermentation peptide prepared in Example 1, 40 parts of guava extract prepared in Preparation Example 1, and 15 parts of PQQ.

[0153] The preparation method is as follows: the raw materials are physically mixed to obtain the product.

[0154] Application Example 2

[0155] This application example provides a composition with anti-glycation and anti-aging effects, which includes, by mass parts, 10 parts of fermented sacha inophyllum protein peptide prepared in Example 2, 60 parts of guava extract prepared in Example 2, and 5 parts of PQQ.

[0156] The preparation method is described in Application Example 1.

[0157] Application Example 3

[0158] This application example provides a composition with anti-glycation and anti-aging effects, which includes, by mass parts, 30 parts of Sacha inca protein fermentation peptide prepared in Example 3, 20 parts of guava extract prepared in Example 3, and 20 parts of PQQ.

[0159] The preparation method is described in Application Example 1.

[0160] Application Example 4-10

[0161] This application example provides a composition with anti-glycation and anti-aging effects. The only difference between this composition and application example 1 is that the guava extract prepared in preparation example 1 is replaced in equal amounts with the guava extracts prepared in preparation examples 4, 5, 6, 7, 8, 9, and 10, while other components and contents remain unchanged.

[0162] The preparation method is described in Application Example 1.

[0163] Comparative Application Example 1

[0164] This application example provides a composition with anti-glycation and anti-aging effects. The only difference between this composition and Application Example 1 is that it does not contain the fermented sacha inophylline protein peptides prepared in Example 1, and the reduced mass of the sacha inophylline protein peptides is proportionally allocated to the mass of the guava extract and PQQ prepared in Example 1.

[0165] The preparation method is described in Application Example 1.

[0166] Comparative Application Example 2

[0167] This application example provides a composition with anti-glycation and anti-aging effects. The only difference between this composition and Application Example 1 is that it does not contain the guava extract prepared in Preparation Example 1, and the reduced mass of the guava extract is proportionally allocated to the mass of the sacha inoculum protein fermented peptide and PQQ prepared in Example 1.

[0168] The preparation method is described in Application Example 1.

[0169] Comparative Application Example 3

[0170] This application example provides a composition with anti-glycation and anti-aging effects. The only difference between this composition and Application Example 1 is that it does not contain PQQ, and the reduced mass of PQQ is proportionally allocated to the mass of the fermented sacha inca protein peptide prepared in Example 1 and the mass of the guava extract prepared in Example 1.

[0171] The preparation method is described in Application Example 1.

[0172] Test Example 1

[0173] Antioxidant effect test

[0174] DPPH solution was dissolved in anhydrous ethanol (concentration 0.1 mg / mL). Vitamin C (positive control) and sample extract solutions were prepared simultaneously (concentration 10 mg / mL for Examples 1-11 and Comparative Examples 1-4, concentration 0.25 mg / mL for Application Examples 1-10 and Comparative Application Examples 1-3). 150 μL of DPPH ethanol solution and 150 μL of extract solution were mixed thoroughly. A blank control group was set up. The mixture was reacted at room temperature in the dark for 30 min, shaken well, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rate of the sample was calculated using the following formula:

[0175]

[0176] Sample group: 150 μL sample solution + 150 μL DPPH alcohol solution

[0177] Sample blank group: 150 μL sample solution + 150 μL anhydrous ethanol

[0178] Control group: 150 μL of sample solvent + 150 μL of DPPH alcohol solution

[0179] Control group: 150 μL sample solvent + 150 μL anhydrous ethanol

[0180] The DPPH free radical scavenging rate (%) results are shown in Table 1.

[0181] Table 1

[0182]

[0183] As shown in Table 1, the fermented sacha inchi protein peptides prepared by this invention and the compositions containing fermented sacha inchi protein peptides both have good antioxidant effects. Guava extract, fermented sacha inchi protein peptides and PQQ have a certain synergistic effect on the above effects. The preparation method of guava extract and fermented sacha inchi protein peptides will affect the above effects.

[0184] Test Example 2

[0185] Anti-glycation test

[0186] The sample solutions were prepared using phosphate buffer (50 mmol / L, pH=7.4) (10 mg / mL for Examples 1-11 and Comparative Examples 1-4, and 0.25 mg / mL for Application Examples 1-10 and Comparative Application Examples 1-3). 1 mL of the sample, bovine serum albumin (BSA, 0.8 mg / mL) solution, glucose solution (200 mM), and phosphate buffer (PBS) were added to test tubes, and the tubes were heated at 60 °C for 24 h. Aminoguanidine hydrochloride was used as a positive control. Fluorescent AGEs were measured using a fluorescence microplate reader with an excitation wavelength of 370 nm and an emission wavelength of 420 nm. The fluorescence intensity (AU) of the AGEs was expressed as a fluorescence intensity.

[0187] Table 2

[0188]

[0189] The inhibition rate of fluorescent AGEs formation by the sample is calculated as follows:

[0190]

[0191] Each group was measured an average of 3 times, and the average value was taken to calculate the AGEs formation inhibition rate. The results are shown in Table 3.

[0192] Table 3

[0193]

[0194] As shown in Table 3, the fermented sacha inchi protein peptides prepared by this invention and the compositions containing fermented sacha inchi protein peptides both have good anti-glycation effects. Guava extract, fermented sacha inchi protein peptides and PQQ have a certain synergistic effect on the above effects. The preparation methods of guava extract and fermented sacha inchi protein peptides will affect the above effects.

[0195] Test Example 3

[0196] Collagenase activity inhibition assay

[0197] Experimental methods:

[0198] The test sample solutions were prepared using Tris-HCl buffer (pH = 7.5 ± 0.2, containing 5 mmol / mL CaCl2) (the concentration was 10 mg / mL for Examples 1-11 and Comparative Examples 1-4, and 0.25 mg / mL for Application Examples 1-10 and Comparative Application Examples 1-3), with tetracycline hydrochloride as a positive control. Blank, control, sample, and sample-blank groups were set up. 60 mL of the test sample solution was added to each well of a 96-well plate. L, blank group uses 60 Replace the sample solution with L buffer. Add 140 μL of enzyme solution (0.2 mg / mL) to the control group and sample group, and add the same volume of buffer to the blank group and sample blank group. After incubating at 37 ℃ for 20 min, add 40 μL of substrate FALGPA solution (0.5 mg / mL) to each group. After incubating at 37 ℃ for 20 min, measure the change in absorbance of each component at 330 nm. Each group was measured three times on average, and the average value was taken. Calculate the inhibition rate of collagenase according to the following formula. The results are shown in Table 4.

[0199]

[0200] Sample blank group: 60 μL sample solution + 140 μL buffer salt + 40 μL substrate

[0201] Sample group: 60 μL sample solution + 140 μL collagenase + 40 μL substrate

[0202] Control group: 60 μL buffer salt + 140 μL collagenase + 40 μL substrate

[0203] Control group: 200 μL buffer + 40 μL substrate

[0204] Table 4

[0205]

[0206] As shown in Table 4, the fermented sacha inchi protein peptides prepared by this invention and the compositions containing fermented sacha inchi protein peptides both have good collagenase inhibition effects. Guava extract, fermented sacha inchi protein peptides and PQQ have a certain synergistic effect on the above effects. The preparation methods of guava extract and fermented sacha inchi protein peptides will affect the above effects.

[0207] Test Example 4

[0208] Fe 2+ Chelation capacity test

[0209] The Fenton reaction involves the reaction of iron ions with hydrogen peroxide under acidic conditions to generate hydroxyl radicals. These radicals accelerate the decomposition of polysaccharides during saccharification. If plant extracts possess metal chelating capabilities, they can bind to iron ions, inhibiting the generation of hydroxyl radicals and thus mitigating the accelerating effect of the Fenton reaction on saccharification. Based on phenoxybenzidine and... The chelating ability of iron ions was determined by a colorimetric reaction, with EDTA-2Na used as a positive control.

[0210] Two mL of sample solutions (concentration 10 mg / mL) from Application Example 1, Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3 were mixed with 2.7 mL of distilled water and 0.1 mL of FeCl₂·4H₂O solution (2 mmol / L). Then, 0.2 mL of phenoxylate (5 mmol / L) was added. After reacting for 10 min at room temperature, the absorbance was measured at 562 nm. Simultaneously, ultrapure water was used as a control group instead of the sample, and ultrapure water was used as a blank control group instead of phenoxylate. 2+ The chelation rate is calculated using the following formula, and the result is as follows: Figure 1 As shown.

[0211]

[0212] In the formula:

[0213] A sample : Sample group absorbance measurement

[0214] A sampleblank : Absorbance determination of blank sample group

[0215] A control : Absorbance measured in the control group

[0216] A controlblank : The absorbance of the control group was measured

[0217] Fe 2+ Fe is a highly reactive transition-state metal ion that can catalyze and accelerate protein glycosylation. Therefore, by chelating Fe... 2+ This is an important pathway to inhibit the formation of AGEs. For example... Figure 1 As shown, Application Example 1, Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3 all affect Fe 2+ It has chelating ability, and the Fe in Example 1 is used. 2+ The chelating ability was significantly better than that of Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3. P<0.001), indicating that the composition of Application Example 1 has a stronger activity advantage in inhibiting AGEs formation. The above data demonstrate that the composition of the present invention can effectively chelate Fe... 2+ It significantly reduces protein glycosylation levels and effectively inhibits the formation of AGEs.

[0218] Test Example 5

[0219] MGO / GO Capture Ability Test

[0220] Principle: Since dicarbonyl compounds glyoxal (GO) and methylglyoxal (MGO) are inherently unstable and do not absorb ultraviolet light at wavelengths of 200–400 nm, dicarbonyl compounds are derivatized to form stable quinoxaline derivatives, which are then detected.

[0221] The method is as follows: MGO / GO (2.2 mmol / L) and different samples (10 mg / mL) were incubated in the same volume in 50 mmol / L phosphate-buffered saline PBS (pH 7.0) at 37 °C for 6 h. 0.5 mL of derivatization reagent OPD (10 mmol / L) was mixed with 0.5 mL of the above incubated sample and reacted at 37 °C for 0.5 h. After passing through a 0.22 μm membrane, the generated MGO / GO derivative was detected by HPLC-DAD.

[0222] HPLC conditions: Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm), injection volume 10 μL, flow rate 1 mL / min, column temperature 30 ℃, mobile phase: aqueous solution (A) containing 0.1% formic acid and methanol (B), gradient elution program: 0–20 min, 30%–70% B. Detection wavelength: 315 nm.

[0223] The formula for calculating the MGO / GO capture rate (%) is as follows:

[0224]

[0225] In the formula, F0 is the peak area of ​​MGO / GO in the control group, and F1 is the peak area of ​​MGO / GO in the sample group.

[0226] like Figure 2 As shown, Application Example 1, Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3 can all effectively capture MGO and GO, and the capture capability of Application Example 1 is significantly better than that of Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3. P <0.01), indicating that the composition of the present invention can inhibit the formation of AGEs by capturing MGO and GO.

[0227] Test Example 6

[0228] Anti-aging effect test

[0229] HFF cells in logarithmic growth phase with good morphology were seeded in 6-well plates at a density of 2.5 × 10⁻⁶ cells / well. 5 Cells / wells were incubated in an incubator for 24 h. Control and sample groups were set up, with three replicates per group. Cells were treated with sample solution (250 μg / mL) for 4 h, followed by induction treatment with H2O2 (1.4 mmol / L) for 2 h. Cells were then re-incubated at 37 ℃ in a 5% CO2 incubator for another 24 h. RNA extraction, reverse transcription, and other procedures were then performed. q PCR analysis was used to determine the relative mRNA expression levels of MMP1, MMP3, and MMP9.

[0230] like Figure 3 As shown, Application Example 1, Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3 all significantly downregulated the expression levels of MMP1, MMP3, and MMP9 genes, and the downregulation ability of Application Example 1 was significantly better than that of Comparative Application Example 1, Comparative Application Example 2, and Comparative Application Example 3. P <0.01), indicating that the composition of the present invention has a certain anti-aging effect.

[0231] The applicant declares that this invention illustrates a method for preparing a fermented peptide from *Sapindus mukorossi* protein, a composition containing the fermented peptide, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0232] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0233] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A composition with anti-glycation and anti-aging effects, characterized in that, The composition comprises, by weight, 10-30 parts of sacha infused protein fermentation peptides, 20-60 parts of guava extract, and 5-20 parts of disodium pyrroloquinoline quinone. The sacha indica protein fermentation peptides were prepared by a method comprising the following steps: (1) The sacha inophyllum pulp was mixed with an organic solvent and defatted under subcritical conditions to obtain defatted sacha inophyllum; (2) Mix defatted Sacha in water and perform microjet pretreatment to obtain pretreated Sacha in water; (3) The pretreated Sacha in a series of steps were subjected to ultrasonic extraction and water bath extraction, and the supernatant was collected by centrifugation to obtain Sacha in a series of steps. (4) Mix Sacha inophylline protein with Bacillus subtilis and ferment to obtain fermentation broth; (5) The fermentation broth is subjected to ultrasonic extraction, filtered, and dried to obtain the final product; The Bacillus subtilis ( Bacillus subtilis )for Bacillus subtilis CICC 20030.

2. The composition with anti-glycation and anti-aging effects according to claim 1, characterized in that, The subcritical temperature is 40-50℃, the pressure is 0.4-0.5 MPa, and the time is 40-60 min; The microjet is subjected to a pressure of 100-300 MPa, a temperature of 50-60 ℃, and 2-6 cycles. Step (3) also includes adjusting the pH to 4-4.6 before sedimentation.

3. The composition with anti-glycation and anti-aging effects according to claim 1, characterized in that, The temperature of the ultrasound in step (3) is 40-60 ℃, the time is 30-60 min, and the power is 400-800 W.

4. The composition with anti-glycation and anti-aging effects according to claim 1, characterized in that, The water bath extraction in step (3) is performed at a temperature of 45-55 ℃ for 30-60 min.

5. The composition with anti-glycation and anti-aging effects according to claim 1, characterized in that, The volume percentage of Bacillus subtilis culture in the fermentation system is 0.5-3%.

6. The composition with anti-glycation and anti-aging effects according to claim 5, characterized in that, The viable count of the Bacillus subtilis culture was 1 × 10⁻⁶. 7 -5×10 7 CFU / mL.

7. The composition with anti-glycation and anti-aging effects according to claim 1, characterized in that, The guava extract is prepared by a method comprising the following steps: (1) Guava was pretreated by steam explosion to obtain pretreated guava; (2) Mix the pretreated guava with water, heat and reflux to extract, filter, concentrate and dry to obtain the product.

8. The composition with anti-glycation and anti-aging effects according to claim 7, characterized in that, The guava in step (1) has a water content of 15-30 wt%; The steam explosion is performed at a pressure of 0.2-0.5 MPa, a temperature of 130-160 ℃, and a time of 0.5-2 min.

9. The use of the composition with anti-glycation and anti-aging effects according to any one of claims 1-8 in the preparation of cosmetic products with anti-glycation, anti-oxidation or anti-aging effects.

Images