A giant kelp oligopeptide, a preparation method thereof and application thereof in cosmetics

Abstract

The application discloses a method for preparing a giant kelp oligopeptide, comprising the following steps: performing ultrasonic treatment on giant kelly particles dispersed in a solvent to obtain a mixed sample; performing supercritical carbon dioxide fluid extraction on the mixed sample in the presence of an entraining agent to obtain an extraction liquid; and performing enzymolysis on the extraction liquid to obtain the giant kelp oligopeptide. The application further discloses the giant kelp oligopeptide prepared by the above method and a cosmetic composition containing the giant kelp oligopeptide. The method of the application can prepare the giant kelp oligopeptide at a high yield, and the prepared giant kelp oligopeptide can penetrate into the skin dermis, provide deep moisturizing, nourish and repair the skin, delay aging, promote the absorption of other components in the cosmetic composition, and further synergistically moisturize and whiten the skin together with double-split yeast.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and relates to a giant kelp oligopeptide, its preparation method, and its application in cosmetics. Background Technology

[0002] Seaweed has a wide range of uses due to its unique composition and functionality. Seaweed contains a certain amount of protein, a relatively large amount of polysaccharides and fiber, a small amount of fat, and abundant inorganic salts and vitamins. The minerals contained in seaweed include calcium, iron, sodium, magnesium, phosphorus, and iodine. Modern science believes that regular consumption of seaweed can effectively regulate blood pH, preventing excessive consumption of alkaline elements (calcium, zinc) due to acid neutralization. Seaweed can selectively remove heavy metal carcinogens such as mercury, cadmium, and lead. Women often suffer from iron-deficiency anemia due to physiological reasons; consuming more seaweed can effectively supplement iron. Regular consumption of seaweed can also make dry skin shiny and improve sebum secretion in oily skin. The abundant vitamins in seaweed can maintain the healthy growth of epithelial tissues and reduce pigmentation spots. Because seaweed is rich in various bioactive substances, such as protein, polysaccharides, highly unsaturated fatty acids, taurine, carotenoids, sterols, and laminine, whether used as a daily food or extracted for medicinal purposes, seaweed contains extremely high-level nutrients for human life. Seaweed protein is an important component of seaweed biological structure, a major raw material for tissue renewal and repair, and has a variety of physiological functions.

[0003] Chinese patent CN105192722A discloses an oligopeptide composition obtained by enzymatic hydrolysis of shellfish or seaweed under ultrasonic treatment. The enzymatic hydrolysis conditions are: ultrasonic power 250-500W, enzyme concentration 2-10 mg / g, substrate mass percentage concentration 2-10% (based on protein content), temperature 45-60℃, and time 1-10 hours. The enzyme includes flavor protease and one or more selected from complex protease, neutral protease, papain, alkaline protease, and trypsin. This oligopeptide composition has high angiotensin-converting enzyme inhibitory activity and a unique umami flavor. The resulting umami seasoning has a blood pressure-lowering health function and a unique flavor.

[0004] Chinese patent CN108203729A discloses a method for preparing giant kelp antioxidant peptides through fermentation. Specifically, it employs a liquid fermentation process, where giant kelp is treated and then fermented with a mixed strain of Bacillus subtilis, Bacillus licheniformis, and Aspergillus oryzae to purify and obtain the giant kelp antioxidant peptides. The prepared antioxidant peptides can be used in cosmetics to scavenge free radicals, enhance the skin's antioxidant capacity, and achieve anti-aging effects.

[0005] Currently, there is still a need to develop effective processing methods for giant kelp so that the active substances in giant kelp can be used as deep moisturizing and antioxidant ingredients in skin care or cosmetic products. Summary of the Invention

[0006] This invention discloses a method for preparing giant kelp oligopeptides and their application in cosmetics. The method of this invention enables the high-yield acquisition of the effective active ingredient from giant kelp: giant kelp oligopeptides.

[0007] The first aspect of the present invention provides a method for preparing giant kelp oligopeptides, the method comprising the following steps:

[0008] (1) Ultrasonic treatment was performed on the giant kelp particles dispersed in the solvent to obtain a mixed sample;

[0009] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction in the presence of an entrainer to obtain an extract;

[0010] (3) The extract was enzymatically hydrolyzed to obtain an enzymatic hydrolysate containing giant kelp oligopeptides.

[0011] In a first aspect of the invention, as a preferred embodiment, giant kelp oligopeptides are prepared using green algae, red algae, and / or brown algae from giant kelp.

[0012] In a first aspect of the invention, as a more preferred embodiment, giant kelp oligopeptides are prepared using green and / or red algae from giant kelp.

[0013] In a first aspect of the invention, as a preferred embodiment, water, ethanol, or propylene glycol is used as a solvent.

[0014] In a first aspect of the invention, as a more preferred embodiment, water is used as a solvent.

[0015] In a first aspect of the invention, as a preferred embodiment, in step (1), the kelp particles are kelp powder that has passed through a 50-100 mesh sieve, for example, kelp powder that has passed through a 50-mesh sieve, a 60-mesh sieve, a 70-mesh sieve, an 80-mesh sieve, a 90-mesh sieve or a 100-mesh sieve.

[0016] In a first aspect of the invention, as a preferred embodiment, the giant kelp particles are obtained by drying and pulverizing fresh giant kelp.

[0017] In this invention, the method for drying fresh giant kelp can be natural air drying or drying using known drying equipment, such as hot air drying, infrared drying, far-infrared drying, microwave drying, freeze drying, etc.

[0018] In a first aspect of the invention, as a preferred embodiment, during the preparation of giant kelp particles, fresh giant kelp is dried at 40-100°C, for example, at 40°C, 45°C, 50°C, 55°C, 60°C, 70°C, 80°C, 90°C, or 95°C. However, to avoid possible deactivation of the giant kelp components, a drying temperature of 40-80°C is preferred, and more preferably 40-60°C.

[0019] In a first aspect of the invention, as a preferred embodiment, during the preparation of giant kelp particles, the fresh giant kelp is first washed with water before drying to remove surface impurities and soluble salts.

[0020] In a first aspect of the invention, the giant kelp particles in step (1) may also be fresh giant kelp that has been finely crushed.

[0021] In a first aspect of the invention, as a preferred embodiment, in step (1), the power of the ultrasonic treatment is 100-500W, for example, the power of the ultrasonic treatment is 100W, 150W, 200W, 250W, 300W, 350W, 400W, 450W or 500W.

[0022] In a first aspect of the invention, as a preferred embodiment, in step (1), the temperature of the ultrasonic treatment is 20-40°C, for example, the temperature of the ultrasonic treatment is 20°C, 25°C, 30°C, 35°C or 40°C.

[0023] In a first aspect of the invention, as a preferred embodiment, in step (1), the ultrasonic treatment time is 30-60 min, for example, the ultrasonic treatment time is 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0024] In a first aspect of the invention, as a more preferred embodiment, in step (1), water is used as a solvent, the ultrasonic treatment power is 100-500W, the ultrasonic treatment temperature is 20-40°C, and the ultrasonic treatment time is 30-60min.

[0025] In a first aspect of the invention, as a particularly preferred embodiment, in step (1), water is used as a solvent, the ultrasonic treatment power is 500W, the ultrasonic treatment temperature is 30°C, and the ultrasonic treatment time is 60min.

[0026] In a first aspect of the invention, as a preferred embodiment, in step (2), the mixed sample is subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1, and residue C1 is subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extract A1 and extract A2 are combined to obtain extract, which is used in step (3).

[0027] In a first aspect of the invention, as a preferred embodiment, in step (2), a polar solvent available in cosmetics is used as an entrainer.

[0028] In a first aspect of the invention, as a more preferred embodiment, in step (2), the entrainer is selected from one or more of water, ethanol, propylene glycol and n-butanol.

[0029] In a first aspect of the invention, as a preferred embodiment, in step (2), the extraction temperature is 25-50°C, for example, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C.

[0030] In a first aspect of the invention, as a preferred embodiment, in step (2), the extraction pressure is 30-50 MPa, for example, the extraction pressure is 30 MPa, 35 MPa, 40 MPa, 45 MPa or 50 MPa.

[0031] In a first aspect of the invention, as a preferred embodiment, in step (2), the extraction time is 2-3 hours each time, for example, 2 hours, 2.5 hours or 3 hours.

[0032] In a first aspect of the invention, as a preferred embodiment, in step (2), the flow rate of carbon dioxide is 500-950 kg / h, for example, the flow rate of carbon dioxide is 500 kg / h, 550 kg / h, 600 kg / h, 650 kg / h, 700 kg / h, 750 kg / h, 800 kg / h, 850 kg / h, 900 kg / h or 950 kg / h.

[0033] In a first aspect of the invention, as a preferred embodiment, in step (3), enzymatic hydrolysis is performed using a proteolytic enzyme.

[0034] In a first aspect of the invention, as a more preferred embodiment, in step (3), the proteolytic enzyme is selected from at least one of trypsin, serine protease, thiol protease, and aspartic protease.

[0035] In a first aspect of the invention, as a particularly preferred embodiment, in step (3), the proteolytic enzyme is a combination of trypsin and thiol protease.

[0036] In a first aspect of the invention, as a preferred embodiment, in step (3), the weight ratio of enzyme to extract is from 1:1,000,000 to 5:1,000,000, for example, the weight ratio of enzyme to extract is 1:1,000,000, 2:1,000,000, 3:1,000,000, 4:1,000,000, or 5:1,000,000. In the expression "weight ratio of enzyme to extract," the weight of the enzyme refers to the sum of the weights of all enzymes used for enzymatic hydrolysis in step (3).

[0037] In a first aspect of the invention, as a preferred embodiment, in step (3), the enzymatic hydrolysis temperature is 30-55°C, for example, 30°C, 35°C, 40°C, 45°C, 50°C or 55°C.

[0038] In a first aspect of the invention, as a preferred embodiment, in step (3), the enzymatic hydrolysis time is 1-3 hours, for example, 1 hour, 2 hours or 3 hours.

[0039] In a first aspect of the present invention, as a preferred embodiment, step (3) further includes terminating the enzymatic hydrolysis reaction after enzymatic hydrolysis; the termination of the enzymatic hydrolysis reaction can be achieved by using high temperature to inactivate the enzyme, for example, by heating the hydrolyzed solution at 85-95°C for 3-10 minutes to terminate the enzymatic hydrolysis.

[0040] In a first aspect of the invention, as a preferred embodiment, the method further includes the step of separating the giant kelp oligopeptides from the enzymatic hydrolysate containing giant kelp oligopeptides, comprising obtaining a mixed peptide filtrate with a relative molecular mass of 200-1000 by filtration, and then freeze-drying the filtrate to obtain the giant kelp oligopeptides. More preferably, the filtration is ultrafiltration and / or nanofiltration. More specifically, activated carbon powder is added to the enzymatic hydrolysate, the enzymatic hydrolysis is terminated by incubating in a boiling water bath for 5 minutes, the enzymatic hydrolysate is cooled to room temperature, and the mixed peptides with a relative molecular mass of 200-1000 are collected by ultrafiltration and nanofiltration, and the filtrate is freeze-dried to obtain the giant kelp oligopeptides.

[0041] The various features in the method for preparing giant kelp oligopeptides in the first aspect of the present invention can be freely combined to form new technical solutions when appropriate.

[0042] The second aspect of the present invention provides a giant kelp oligopeptide, which is prepared according to the first aspect of the present invention, namely, the method for preparing giant kelp oligopeptides.

[0043] The giant kelp oligopeptides prepared by the method of the present invention are mixed peptides with a relative molecular mass of 200-1000.

[0044] The giant kelp oligopeptides prepared by the method of this invention can activate skin cells, promote the division of epidermal basal cells, penetrate deep into the skin base, deeply hydrate, moisturize and repair the skin, and delay aging.

[0045] Furthermore, the inventors discovered that the giant kelp oligopeptides prepared by the method of this invention can synergistically replenish and lock in moisture with double bifida ferment lysate, thus achieving a synergistic whitening and moisturizing effect. Moreover, the giant kelp oligopeptides can promote the absorption of other ingredients, making each component in the cosmetic composition more effective and increasing the utilization rate of each component, resulting in better effects such as hydration, moisturizing, and skin repair.

[0046] A third aspect of the invention provides a composition comprising giant kelp oligopeptide, hyaluronic acid, Centella asiatica extract, bifida ferment lysate, nicotinamide, and sodium pyrrolidone carboxylate, wherein the giant kelp oligopeptide is prepared by the method for preparing giant kelp oligopeptide according to the first aspect of the invention.

[0047] In a third aspect of the invention, as a preferred embodiment, the composition comprises, by weight percentage, 4-30% of giant kelp oligopeptide, 15-30% of hyaluronic acid, 15-25% of Centella asiatica extract, 5-25% of Bifida ferment lysate, 5-15% of nicotinamide and 5-25% of sodium pyrrolidone carboxylate, wherein the giant kelp oligopeptide is prepared according to the method for preparing giant kelp oligopeptide according to the first aspect of the invention.

[0048] In a third aspect of the invention, as a preferred embodiment, the composition comprises, by weight percentage, 8-30% of giant kelp oligopeptide, 15-30% of hyaluronic acid, 15-20% of Centella asiatica extract, 8-20% of bifida ferment lysate, 8-15% of nicotinamide and 10-25% of sodium pyrrolidone carboxylate, wherein the giant kelp oligopeptide is prepared according to the method for preparing giant kelp oligopeptide according to the first aspect of the invention.

[0049] The compositions of the present invention may also include other commonly used cosmetic ingredients.

[0050] In a third aspect of the invention, as a preferred embodiment, the composition further includes any cosmetic excipient / carrier known in the art.

[0051] In a third aspect of the invention, as a preferred embodiment, the composition optionally further comprises one or more of the following ingredients known in the art as cosmetics: chelating agents, plant extracts, colorants, bleaching agents, emollients, exfoliants, fragrances, humectants, moisturizers, preservatives, skin care agents, skin penetration enhancers, stabilizers, thickeners, viscosity improvers, vitamins, anti-aging agents, wrinkle reducers, skin brighteners, anti-acne agents, and sebum reducers, or any combination thereof.

[0052] In a third aspect of the invention, as a preferred embodiment, the formulation of the composition includes creams, ointments, emulsions, masks, balms, gels, etc.

[0053] In a third aspect of the invention, as a preferred embodiment, the formulation of the composition is selected from: lotions, creams, masks, eye creams, and serums.

[0054] The composition of the present invention has excellent effects in deep hydration and moisturizing, skin repair, and delaying aging.

[0055] The fourth aspect of the present invention provides a method for preparing the composition provided in the third aspect of the present invention, the method comprising: mixing the giant kelp oligopeptide provided in the second aspect of the present invention with hyaluronic acid, Centella asiatica extract, Bifida ferment lysate, nicotinamide and sodium pyrrolidone carboxylate.

[0056] The fifth aspect of the present invention provides the use of the method for preparing giant kelp oligopeptides according to the first aspect of the present invention, the giant kelp oligopeptides provided by the second aspect of the present invention, the composition provided by the third aspect of the present invention, and the method for preparing the composition provided by the fourth aspect of the present invention in the preparation of deep hydrating, moisturizing, and antioxidant preparations.

[0057] Compared with the prior art, the beneficial effects of the present invention are:

[0058] The method of this invention can obtain the effective active ingredient in giant kelp, giant kelp oligopeptide, in a high yield.

[0059] The giant kelp oligopeptide prepared by the method of this invention can activate skin cells, promote the division of epidermal basal cells, penetrate deep into the skin base, deeply hydrate, moisturize and repair the skin, and delay aging.

[0060] The giant kelp oligopeptide prepared by the method of this invention can synergistically replenish and lock in moisture with bifida ferment lysate, and work together to whiten and moisturize. Furthermore, the giant kelp oligopeptide can promote the absorption of other ingredients, making each component in the cosmetic composition more effective and increasing the utilization rate of each component, resulting in better effects such as hydration, moisturization, and skin repair. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of the present invention clearer, exemplary embodiments of the present invention will be described below. The following embodiments are for illustrative purposes only and not for limiting the present invention in any way; the scope of protection of the present invention includes, but is not limited to, the following embodiments.

[0062] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0063] The various proteases used in this invention, calculated on a dried basis, shall have an activity of not less than 50,000 units per 1 mg. The giant kelp used in the following examples is a brown algae.

[0064] The term "giant kelp" as used in this article refers to giant or large seaweed, which are multicellular algae that attach to rocks or other underwater substrates using holdfasts, and can reach a maximum length of over 1 meter. Giant kelp (including green, red, and brown algae) has a high content of active proteins, especially in green and red algae, where the content is much higher than in brown algae.

[0065] The giant kelp used in this invention is derived from wild deep-sea giant kelp or artificially cultivated giant kelp.

[0066] The term “oligopeptide” as used in this article has the common meaning in the field. Oligopeptides, also known as oligopeptides, are small molecule active peptides formed by 2-10 amino acids linked by peptide bonds. They are characterized by small molecular weight, easy absorption, and high nutritional value.

[0067] The term "giant kelp oligopeptide" as used in this article refers to oligopeptides obtained from giant kelp.

[0068] As used herein, the term "ultrasonic treatment" has its common meaning in the art, referring to a technique that uses the energy of ultrasound to act on a substance to change its properties and / or state. In the method described above in this invention, ultrasonic treatment initially dissolves the water-soluble proteins in giant kelp and facilitates their uniform dispersion.

[0069] The term "supercritical carbon dioxide fluid extraction" as used herein has its conventional meaning in the art. A supercritical fluid is a state of matter that is neither gaseous nor liquid; it exists only when its temperature and pressure exceed a critical point. Supercritical fluids have a high density, similar to liquids, while their viscosity is closer to that of gases. Therefore, supercritical fluids are ideal extractants. Furthermore, optimal mixing ratios can be obtained by controlling the conditions, and then the supercritical fluid can be converted into a normal gas by reducing pressure and increasing temperature, causing the extracted substances to completely or substantially precipitate, thus achieving separation and purification. Therefore, the supercritical carbon dioxide fluid extraction process is a combination of extraction and separation processes. In the method of this invention, supercritical carbon dioxide fluid extraction further extracts active substances such as proteins, amino acids, and unsaturated fatty acids, which serve as raw materials for the next enzymatic hydrolysis step.

[0070] As used herein, the term "entrainer" refers to an entrainer added during supercritical carbon dioxide fluid extraction to promote the extraction of polar substances. In the method described above, an entrainer is added during supercritical carbon dioxide fluid extraction to enhance the extraction effect.

[0071] As used herein, the term "enzymatic hydrolysis" has its common meaning in the art and refers to decomposition catalyzed by enzymes. In the method described above in this invention, the enzymatic hydrolysis process decomposes macromolecules in the extract that can be decomposed by enzymes into smaller molecules.

[0072] In this article, the term "mesh" as used in expressions such as "50-mesh sieve," "80-mesh sieve," and "100-mesh sieve" refers to the number of sieve openings per inch (1 inch equals 2.54 centimeters) of sieve length. For example, a 50-mesh sieve means that the sieve has an average of about 50 openings per inch of sieve length.

[0073] As used herein, the term "composition" preferably refers to a cosmetic composition, intended to describe compositions for topical application to human skin, including leave-on and wash-off products. The term "skin" as used herein includes the skin on the face, neck, chest, back, arms, hands, legs, and scalp.

[0074] The composition of this invention contains hyaluronic acid, also known as hyaluronic acid, with the molecular formula (C... 14 H 21 NO 11 ) n It is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine.

[0075] The composition of this invention contains Centella asiatica extract, the main component of which is asiaticoside. It has scar-reducing effects, promotes the vitality of scar fibroblasts, and synthesizes and secretes collagen fibers (with collagen as the main component) and extracellular matrix, playing an important role in filling wound defects. It also has whitening and spot-removing effects, as well as antibacterial and anti-inflammatory effects. As a skin conditioning agent, it enables cosmetics to achieve their moisturizing, whitening, anti-aging, and acne-removing effects.

[0076] The composition of the present invention contains Bifida Ferment Lysate, scientifically known as Bifidobacterium, which can significantly increase the content and biological activity of superoxide dismutase in the blood, promote the scavenging of free radicals in the body, inhibit plasma lipid peroxidation, and delay the aging of the body; it also has the effects of moisturizing and oil control, shrinking pores, repairing and whitening, and soothing and calming.

[0077] The composition of this invention contains nicotinamide, also known as nicotinamide, which is an amide compound of nicotinic acid. It participates in the body's metabolic processes and is used to prevent and treat nicotinic acid deficiency diseases such as pellagra, and to improve uneven skin tone. It is a white crystalline powder; odorless or almost odorless, with a bitter taste; slightly hygroscopic. It is readily soluble in water or ethanol and soluble in glycerol.

[0078] The composition of this invention contains sodium pyrrolidone carboxylate, abbreviated as PCA-Na. It is an important component of the body's natural moisturizing factor, with high hygroscopicity, non-toxicity, non-irritation, and good stability. It is an ideal natural cosmetic and health product for modern skin and hair care, which can make the skin and hair moisturized, soft, elastic, shiny, and antistatic, and can effectively replenish and lock in moisture.

[0079] In the following composition examples of the present invention, all other components in the composition except for giant kelp oligopeptide are commercially available. Hyaluronic acid is available from Sigma-Aldrich (Shanghai) Trading Co., Ltd., Centella asiatica extract is available from Xi'an Ruierli Biotechnology Co., Ltd., Bifida ferment lysate is available from Shandong Hemantang Biotechnology Co., Ltd., nicotinamide is available from Wuhan Deyuancheng Biotechnology Co., Ltd. or Jiangsu Caiwei Biotechnology Co., Ltd., and sodium pyrrolidone carboxylate is available from Wuhan Huaxiang Kejie Biotechnology Co., Ltd.

[0080] Example 1: Preparation of giant kelp oligopeptides

[0081] Example 1.1

[0082] Giant kelp oligopeptide A1 was prepared according to the following steps:

[0083] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an oven at 60°C, then pulverized with a pulverizer and passed through a 100-mesh sieve. The sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 400W, the temperature was 30°C, and the time was 60min to obtain a mixed sample.

[0084] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 was subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extracts A1 and A2 were combined. 5g of water was added as an entrainer during extraction. The extraction temperature was 50℃, the extraction pressure was 45MPa, the extraction time was 3h each time, and the carbon dioxide flow rate was 800kg / h to obtain the extract.

[0085] (3) The extract was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzyme to the weight of extract was 4:1,000,000. The hydrolysis temperature was 40℃ and the hydrolysis time was 3 hours. After the hydrolysis was completed, the enzyme was inactivated by heating at 90℃ for 8 minutes. The hydrolysate was cooled to room temperature and then added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide A1.

[0086] Example 1.2

[0087] Prepare giant kelp oligopeptide A2 according to the following steps:

[0088] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an oven at 60°C, then pulverized with a pulverizer and passed through a 50-mesh sieve. The sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 200W, the temperature was 20°C, and the time was 30min to obtain a mixed sample.

[0089] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 was subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extracts A1 and A2 were combined. 2g of water was added as an entrainer during extraction. The extraction temperature was 30℃, the extraction pressure was 30MPa, the extraction time was 2h, and the carbon dioxide flow rate was 500kg / h to obtain the extract.

[0090] (3) The extract was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzyme to the weight of extract was 5:1,000,000. The hydrolysis temperature was 35℃ and the hydrolysis time was 3 hours. After the hydrolysis was completed, the enzyme was inactivated by heating at 90℃ for 8 minutes and then cooled to room temperature. The hydrolysate was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide A2.

[0091] Example 1.3

[0092] Prepare giant kelp oligopeptide A3 according to the following steps:

[0093] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an oven at 60°C, then pulverized with a pulverizer and passed through a 100-mesh sieve. The sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 400W, the temperature was 20°C, and the time was 30min to obtain a mixed sample.

[0094] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 was subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extracts A1 and A2 were combined. 4g of water was added as an entrainer during extraction. The extraction temperature was 40℃, the extraction pressure was 40MPa, the extraction time was 2.5h, and the carbon dioxide flow rate was 750kg / h to obtain the extract.

[0095] (3) The extract was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzyme to the weight of extract was 4:1,000,000. The hydrolysis temperature was 40℃ and the hydrolysis time was 3 hours. After the hydrolysis was completed, the enzyme was inactivated by heating at 90℃ for 8 minutes and then cooled to room temperature. The hydrolysate was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide A3.

[0096] Example 1.4

[0097] Prepare giant kelp oligopeptide A4 according to the following steps:

[0098] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an oven at 60°C, then pulverized with a pulverizer and passed through a 100-mesh sieve. The sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 500W, the temperature was 30°C, and the time was 60min to obtain a mixed sample.

[0099] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 was subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extracts A1 and A2 were combined. 5g of water was added as an entrainer during extraction. The extraction temperature was 40℃, the extraction pressure was 45MPa, the extraction time was 2.5h, and the carbon dioxide flow rate was 800kg / h to obtain the extract.

[0100] (3) The extract was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzyme to the weight of extract was 4:1,000,000. The hydrolysis temperature was 40℃ and the hydrolysis time was 3 hours. After the hydrolysis was completed, the enzyme was inactivated by heating at 90℃ for 8 minutes and then cooled to room temperature. The hydrolysate was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide A4.

[0101] Example 1.5

[0102] Except for the extraction conditions in step (2), which are different from those in Example 1.1, the other steps are the same as in Example 1.1. The extraction conditions in this example are: extraction temperature of 30°C, extraction pressure of 50 MPa, extraction time of 3 h each time, carbon dioxide flow rate of 900 kg / h, and the resulting extract. The giant kelp oligopeptide obtained in this example is A5.

[0103] Comparative Example 1.1

[0104] The method for preparing giant kelp oligopeptides in Comparative Example 1.1 was the same as in Example 1.1, except that the giant kelp particles were not subjected to ultrasonic treatment. The giant kelp oligopeptides prepared in this comparative example were designated as D1. Specifically, giant kelp oligopeptide D1 was prepared according to the following steps:

[0105] (1) Wash the freshly harvested giant kelp with water to remove surface impurities and soluble salts, dry it in an oven at 60°C, then crush it with a pulverizer, pass it through a 100-mesh sieve, and take the sieve material to obtain giant kelp powder.

[0106] (2) Place 15g of giant kelp powder in 1000mL of deionized water and stir evenly. Perform supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. Perform supercritical carbon dioxide fluid extraction on residue C1 to obtain extract A2. Combine extracts A1 and A2. Add 5g of water as an entrainer during extraction. The extraction temperature is 50℃, the extraction pressure is 45MPa, the extraction time is 3h, and the carbon dioxide flow rate is 800kg / h to obtain the extract.

[0107] (3) The extract was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzyme to the weight of extract was 4:1,000,000. The hydrolysis temperature was 40℃ and the hydrolysis time was 3 hours. After the hydrolysis was completed, the enzyme was inactivated by heating at 90℃ for 8 minutes and then cooled to room temperature. The hydrolysate was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide D1.

[0108] Comparative Example 1.2

[0109] The method for preparing giant kelp oligopeptides in Comparative Example 1.2 was the same as in Example 1.1, except that supercritical carbon dioxide fluid extraction was not performed. The giant kelp oligopeptides prepared in this comparative example were designated as D2. Specifically, giant kelp oligopeptide D2 was prepared according to the following steps:

[0110] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an oven at 60°C, then pulverized with a pulverizer and passed through a 100-mesh sieve. The sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 400W, the temperature was 30°C, and the time was 60min to obtain a mixed sample.

[0111] (2) The mixed sample was enzymatically hydrolyzed with trypsin and thiol protease (mass ratio 1:1), with enzyme activities of 430,000 U / g and 350,000 U / g, respectively. The total weight of enzymes to the weight of the mixed sample was 4:1,000,000. The hydrolysis temperature was 40℃. After 3 hours of hydrolysis, the enzymes were inactivated by heating at 90℃ for 8 minutes. After cooling to room temperature, the hydrolysate was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide D2.

[0112] Comparative Example 1.3

[0113] The method for preparing giant kelp oligopeptides in Comparative Example 1.3 was the same as in Example 1.1, except that enzymatic hydrolysis was not performed. The giant kelp oligopeptide prepared in this comparative example was designated as D3. Specifically, giant kelp oligopeptide D3 was prepared according to the following steps:

[0114] (1) Freshly harvested giant kelp was washed with water to remove surface impurities and soluble salts, dried in an 80℃ oven, then pulverized with a pulverizer, passed through a 100-mesh sieve, and the sieve material was collected to obtain giant kelp powder. 15g of giant kelp powder was placed in 1000mL of deionized water for ultrasonic treatment. The ultrasonic treatment power was 400W, the temperature was 30℃, and the time was 60min to obtain a mixed sample.

[0115] (2) The mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 was subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extracts A1 and A2 were combined. 5g of water was added as an entrainer during extraction. The extraction temperature was 50℃, the extraction pressure was 45MPa, the extraction time was 3h, and the carbon dioxide flow rate was 800kg / h to obtain the extract.

[0116] (3) The extract was added to an ultrafiltration and nanofiltration system at an operating pressure of 0.25 MPa. Mixed peptides with a relative molecular mass of 200-1000 were collected by ultrafiltration and nanofiltration. The filtrate was freeze-dried to obtain giant kelp oligopeptide D3.

[0117] Comparative Example 1.4

[0118] Except for the extraction conditions in step (2), which are different from those in Example 1.1, the other steps are the same as in Example 1.1. The extraction conditions in this example are: extraction temperature of 30°C, extraction pressure of 20MPa, extraction time of 1h, and carbon dioxide flow rate of 300 kg / h to obtain the extract; the giant kelp oligopeptide obtained in this comparative example is D4.

[0119] Comparative Example 1.5

[0120] Except for the enzymatic hydrolysis step in step (3), which differs from that in Example 1.1, the other steps are the same as in Example 1.1. The enzymatic hydrolysis steps in this example are as follows: papain and alkaline protease with activities of 290,000 U / g and 380,000 U / ml, respectively, are used in a mass ratio of 1:1. The weight ratio of enzyme to extract is 4:1,000,000. The hydrolysis temperature is 40°C, and the hydrolysis time is 3 hours. The subsequent enzyme inactivation and separation procedures are the same as in Example 1.1. The giant kelp oligopeptide obtained in this comparative example is D5.

[0121] Determination of the yield of giant kelp oligopeptides

[0122] The yield of giant kelp oligopeptides was calculated using the following formula:

[0123]

[0124] The determination of tannic acid-soluble protein content refers to DB35 / T 1089-2011. The protein content in the filtrate after precipitation with 16% tannic acid in the protease hydrolysate is determined by the Kjeldahl method. The result obtained is the tannic acid-soluble protein content.

[0125] The determination of free amino acid content refers to DB35 / T 1089-2011, which uses formaldehyde titration to determine the content of free amino acids in the filtrate after precipitation with 16% tannic acid.

[0126] The total protein content was determined according to GB5009.5-2010, using the Kjeldahl method.

[0127] The yields of the giant kelp oligopeptides prepared in each example and comparative example are shown in Table 1.

[0128] Table 1. Yield of giant kelp oligopeptides

[0129] Giant kelp oligopeptides Yield (%) A1 37.16 A2 31.17 A3 34.23 A4 42.45 A5 40.13 D1 8.8 D2 18.8 D3 1.7 D4 25.7 D5 21.9

[0130] It can be seen that the method according to the present invention can obtain the effective active ingredient in giant kelp, giant kelp oligopeptides, in a high yield. According to the method of the present invention, the giant kelp particles dispersed in a solvent are first subjected to ultrasonic treatment to initially dissolve the water-soluble proteins in the giant kelp. Then, the active substances such as proteins, amino acids, and unsaturated fatty acids are further extracted by supercritical carbon dioxide fluid extraction. Following this, enzymatic hydrolysis is performed, and filtration yields a mixed peptide with a relative molecular mass of 200-1000. Failure to perform ultrasonic treatment on the giant kelp particles, or to perform supercritical carbon dioxide fluid extraction on the mixed sample, or to perform enzymatic hydrolysis of the extract, or to perform the corresponding steps outside the scope defined herein, will result in a lower yield of giant kelp oligopeptides.

[0131] Effect test of giant kelp oligopeptide

[0132] The cell experiment cycle is 2 months:

[0133] ① Epidermal cell culture: Oligopeptide dosage was 0.025 IU / mL; specifically: surgical remnants of skin, selecting those with thin keratinization, were cut into 0.5 square centimeter pieces. Placed in 0.02% EDTA at room temperature for 5 minutes. Then, added 0.25% trypsin and incubated overnight at 4°C. The skin pieces were removed, and the epidermis was separated from the dermis using forceps or tweezers. The epidermis was removed, cut into smaller pieces, and placed in 0.25% trypsin at 37°C for 60 minutes. The mixture was repeatedly pipetted to prepare a suspension. After filtering through an 80-mesh stainless steel mesh, the suspension was centrifuged at low speed, and the supernatant was discarded. Cell suspensions were prepared by directly adding culture medium (Eagle with 20% fetal bovine serum), inoculated into culture flasks, and cultured in a CO2 incubator for 24 hours. The oligopeptides prepared in Examples 1.1 to 1.5 and Comparative Examples 1.1 to 1.5 were then added to different cell culture media in the amounts described above. After 2 months of culture, the number of epidermal cells was measured, and the percentage of epidermal cell proliferation was calculated using the following formula: (Total number of cells after 2 months of culture / Number of cells before the addition of oligopeptides) - 1.

[0134] ② Skin keratinocyte culture: Five circumcision specimens were collected from adults aged 20-30 years (with informed consent from both the patient and their family). The specimens were cut into 10mm × 20mm pieces and digested in 2.5g / L dispase at 4°C for 18 hours. The epidermis was then peeled off and placed in a mixture of 2.5g / L trypsin and 0.2g / L EDTA (volume ratio 1:1), and digested at 37°C for 5 minutes. After digestion was terminated, the cells were collected by pipetting, filtering, and centrifugation. The cells were added to K-SFM medium containing bovine pituitary extract (BPE), and HKC (human renal tubular epithelial cells) were cultured at a concentration of 1 × 10⁻⁶. 5 / cm 2 Inoculate the cells and change the culture medium every 3 days. When the cells reach 80% confluence, passage them, discard the culture medium, add 3 ml of the above-mentioned mixed digestion solution to each culture dish, digest at 37°C for 1 min, discard the digestion solution, and continue digestion with the remaining digestion solution for 8 min. When cells shrink and become rounded under an inverted microscope, and when cells slide down the culture dish like quicksand when tilted, stop digestion with DMEM, centrifuge and wash twice, resuspend the cells in K-SFM, passage at a 1:3 ratio, and add oligopeptide at a dose of 5 mg / kg.

[0135] ③Promoting collagen production: Cell culture is the same as ①, with oligopeptide dosage at 10 ng / ml.

[0136] ④ To promote the water content of the stratum corneum of the skin, the cell culture method is the same as ②, and the oligopeptide dosage is 0.1%.

[0137] The test results are shown in Table 2.

[0138] Table 2: Effects of Giant Kelp Oligopeptides

[0139]

[0140] As can be seen, compared with the comparative example, the giant kelp oligopeptide prepared according to the method of the present invention has a significantly stronger promoting effect on the proliferation of epidermal cells, the production of collagen, the proliferation of stratum corneum cells, and the water content of the stratum corneum.

[0141] Example 2: Preparation of a composition containing giant kelp oligopeptides

[0142] Example 2.1

[0143] Composition Z1 is prepared by mixing 8% kelp oligopeptide, 30% hyaluronic acid, 20% Centella asiatica extract, 20% Bifida ferment lysate, 10% nicotinamide and 12% sodium pyrrolidone carboxylate by weight percentage; wherein the kelp oligopeptide is kelp oligopeptide A4 prepared according to Example 1.4.

[0144] Example 2.2

[0145] Composition Z2 is prepared by mixing 25% kelp oligopeptide, 15% hyaluronic acid, 20% Centella asiatica extract, 8% Bifida ferment lysate, 8% nicotinamide and 24% sodium pyrrolidone carboxylate by weight percentage; wherein the kelp oligopeptide is kelp oligopeptide A4 prepared according to Example 1.4.

[0146] Example 2.3

[0147] Composition Z3 is prepared by mixing 20% ​​by weight of giant kelp oligopeptide, 20% by weight of hyaluronic acid, 20% by weight of centella asiatica extract, 8% by weight of bifida ferment lysate, 8% by weight of nicotinamide and 24% by weight of sodium pyrrolidone carboxylate; wherein the giant kelp oligopeptide is giant kelp oligopeptide A4 prepared according to Example 1.4.

[0148] Example 2.4

[0149] Composition Z4 is prepared by mixing 20% ​​kelp oligopeptide, 30% hyaluronic acid, 15% Centella asiatica extract, 10% Bifida ferment lysate, 15% nicotinamide and 10% sodium pyrrolidone carboxylate by weight percentage; wherein the kelp oligopeptide is kelp oligopeptide A4 prepared according to Example 1.4.

[0150] Comparative Example 2.1

[0151] Composition Z5 is prepared by mixing 0% giant kelp oligopeptide, 30% hyaluronic acid, 15% centella asiatica extract, 30% bifida ferment lysate, 15% nicotinamide and 10% sodium pyrrolidone carboxylate by weight percentage.

[0152] Comparative Example 2.2

[0153] Composition Z6 is prepared by mixing 30% kelp oligopeptide, 30% hyaluronic acid, 15% Centella asiatica extract, 0% Bifida ferment lysate, 15% nicotinamide and 10% sodium pyrrolidone carboxylate by weight percentage; wherein the kelp oligopeptide is kelp oligopeptide A4 prepared according to Example 1.4.

[0154] Skin moisture content test

[0155] Twenty subjects aged 18 to 30 years without skin diseases were randomly selected. Two weeks prior to the experiment, they avoided using any type of skincare, cosmetics, or medications. On the day of the experiment, subjects sat quietly in an environment with a temperature of 25±2℃ and humidity of 50±5% for 30 minutes. Under the guidance of staff, the test areas were cleaned with water. A 5cm × 5cm test area was marked on the inner forearm of both arms. Staff applied a measured amount of product (i.e., test compositions Z1 to Z6) evenly to the right side of the test area. The left side served as a blank control group, with no product applied. During the test, subjects were prohibited from applying any other cosmetics, skincare, or medications. The measurement period was 5 hours, and subjects were prohibited from drinking water, eating, and strenuous exercise. The skin surface was tested using a MultiprobeAdapter System MPA transepidermal water loss analyzer from Courage+Khazaka (Germany) at time points: before application, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after application. Five measurements were taken for each area, and the average value was recorded. The data before application was recorded as T0, and the data after n hours was recorded as T. n The formulas for calculating the rate of change and relative rate of change are as follows:

[0156]

[0157]

[0158] Where: T0—skin background value (%) in the test area before applying cosmetics;

[0159] T n —Skin values ​​(%) after n hours of cosmetic application in the test area;

[0160] T0'—Skin's baseline value (%) before applying cosmetics to the blank area;

[0161] T n '——Skin values ​​after n hours without using cosmetics in the blank area.'

[0162] The test results are shown in Table 3.

[0163] Table 3: Results of Skin Moisture Content Test

[0164]

[0165] As can be seen, compared with the control group, applying the composition according to the present invention significantly increased skin moisture content, even 5 hours after application, the skin moisture content remained significantly higher than that of the control group. This indicates that the composition according to the present invention has a strong moisturizing effect on the skin, effectively replenishing and locking in moisture. Applying compositions without giant kelp oligopeptide (Z5) or without bifida ferment lysate (Z6) resulted in significantly lower skin moisture content 1 to 5 hours after application compared to compositions according to the present invention (e.g., Z4). This indicates that giant kelp oligopeptide can synergistically work with bifida ferment lysate to replenish and lock in moisture, exhibiting a synergistic effect.

[0166] Stability testing of the composition

[0167] The compositions prepared in Examples 2.1 to 2.4 were subjected to heat and cold resistance tests. The samples were coated on the slant surface and placed in environments of 40°C and -7°C for 24 hours respectively. The samples were observed to see if there were any phenomena such as layering or oil-water separation. The samples were also sampled using the 5-point sampling method on the slant surface as described above to observe whether crystals precipitated.

[0168] After heat and cold resistance tests, the samples showed no stratification or oil-water separation, and no crystal precipitation, indicating that the prepared compositions all have good stability.

[0169] The above embodiments are merely illustrative examples and are not intended to limit the implementation. Those skilled in the art will recognize that various variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations; therefore, any obvious variations or modifications derived therefrom remain within the scope of protection of this invention.

Claims

1. A method for preparing giant kelp oligopeptides, characterized in that, The method includes the following steps: (1) Ultrasonic treatment was performed on the giant kelp particles dispersed in the solvent to obtain a mixed sample; (2) In the presence of an entrainer, the mixed sample was subjected to supercritical carbon dioxide fluid extraction to obtain an extract; (3) The extract was enzymatically hydrolyzed to obtain an enzymatic hydrolysate containing giant kelp oligopeptides; The ultrasonic treatment power is 100-500W; the supercritical carbon dioxide fluid extraction temperature is 30-50℃, the extraction pressure is 30-50MPa, the extraction time is 2-3h, and the carbon dioxide flow rate in the supercritical carbon dioxide fluid extraction is 500-950 kg / h; enzymatic hydrolysis is performed using trypsin and thiol protease, with a trypsin to thiol protease mass ratio of 1:

1.

2. The method according to claim 1, characterized in that, The giant kelp particles are derived from green algae, red algae, and / or brown algae; And / or, the giant kelp particles are giant kelp powder that has passed through a 50-100 mesh sieve.

3. The method according to claim 2, characterized in that, The giant kelp particles are giant kelp powder that has passed through a 100-mesh sieve.

4. The method according to claim 1, characterized in that, The method further includes the step of separating giant kelp oligopeptides from an enzymatic hydrolysate containing giant kelp oligopeptides, including obtaining a mixed peptide filtrate with a relative molecular mass of 200-1000 by filtration, and then freeze-drying the filtrate to obtain giant kelp oligopeptides.

5. The method according to claim 4, characterized in that, The filtration is ultrafiltration and / or nanofiltration.

6. The method according to claim 1, characterized in that, The solvent is water, ethanol, or propylene glycol; The entrainer is a polar solvent that can be used in cosmetics.

7. The method according to claim 6, characterized in that, The entrainer is water, ethanol, propylene glycol, or n-butanol.

8. The method according to claim 1, characterized in that, The temperature for ultrasonic treatment is 20-40℃; The ultrasonic treatment time is 30-60 minutes.

9. The method according to claim 8, characterized in that, The ultrasonic treatment power is 500 W; the ultrasonic treatment temperature is 30℃; and the ultrasonic treatment time is 60 min.

10. The method according to claim 1, characterized in that, In step (2), the mixed sample is subjected to supercritical carbon dioxide fluid extraction to obtain extract A1 and residue C1. The residue C1 is subjected to supercritical carbon dioxide fluid extraction to obtain extract A2. Extract A1 and extract A2 are combined to obtain extract.

11. The method according to claim 1, characterized in that, The extraction temperature of the supercritical carbon dioxide fluid extraction is 40°C. The extraction pressure of the supercritical carbon dioxide fluid extraction is 45 MPa; The extraction time for the supercritical carbon dioxide fluid extraction is 2.5 h; The flow rate of carbon dioxide in the supercritical carbon dioxide fluid extraction is 800 kg / h.

12. The method according to any one of claims 1 to 11, characterized in that, The weight ratio of enzyme to extract is 1:1000000 to 5:1000000; The enzymatic hydrolysis temperature is 30-55℃; The enzymatic hydrolysis time is 1-3 hours.

13. The method according to claim 12, characterized in that, The weight ratio of enzyme to extract is 4:1000000; the enzymatic hydrolysis temperature is 40℃; and the enzymatic hydrolysis time is 3 hours.

14. Giant kelp oligopeptides prepared by the method of any one of claims 1 to 13.

15. A composition, characterized in that, The composition comprises, by weight percentage: 4-30% giant kelp oligopeptide, 15-30% hyaluronic acid, 15-25% centella asiatica extract, 5-25% bifida ferment lysate, 5-15% nicotinamide and 5-25% sodium pyrrolidone carboxylate. The giant kelp oligopeptide is prepared by the method described in any one of claims 1 to 13.

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