Method for preparing euphausia superba peptide from lactobacillus plantarum and application of euphausia superba peptide

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a method for preparing euphausia superba peptide from lactobacillus plantarum and application of the euphausia superba peptide. The method for preparing euphausia superba peptide from lactobacillus plantarum comprises the following steps: step S1, degreasing treatment, step S2, sterilization treatment, step S3, fermentation, step S4, enzymolysis and step S5, enzyme deactivation. According to the euphausia superba peptide and the preparation method thereof, the fermentation synergist, the enzymatic promoter and the composite strain are added, and through the synergistic effect, the prepared euphausia superba peptide has the advantages of high yield, low average molecular weight, small irritation and strong moisturizing and water locking performance, and has a wide application prospect in the field of cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a method for preparing Antarctic krill peptides by Lactobacillus plantarum and its application in skin care products. Background Art

[0002] Antarctic krill powder is rich in components such as protein, astaxanthin, and phospholipid fatty acids, and is mostly used for the extraction of krill oil. The defatted krill powder after oil extraction contains a large amount of high-quality protein, and the preparation of krill peptides with functional activities such as antioxidant and blood pressure lowering has attracted the attention of the industry.

[0003] In the prior art, Bacillus subtilis is widely used in the fermentation extraction process of Antarctic krill peptides due to its rich enzyme production and rapid growth. However, in the above extraction process, Bacillus subtilis has problems such as a long fermentation time, limited efficiency, and a decrease in enzyme production ability due to cell autolysis, ultimately resulting in a decrease in the yield of Antarctic krill peptides. At the same time, the average molecular weight range of Antarctic krill peptides obtained by this method is relatively large, and the antioxidant effect is average, which cannot meet the requirements of skin care products for moisturizing and water locking. Summary of the Invention

[0004] To solve the problems mentioned in the above background art, the present invention provides a method for preparing Antarctic krill peptides by Lactobacillus plantarum and its application in skin care products.

[0005] In the first aspect, the present invention provides a method for preparing Antarctic krill peptides by Lactobacillus plantarum, adopting the following technical scheme: A method for preparing Antarctic krill peptides by Lactobacillus plantarum specifically includes the following steps: Step S1, degreasing treatment: According to a material-liquid ratio of 1:12 - 16, add Antarctic krill powder to an ethanol aqueous solution, stir for 6.2 - 8.4 h, stand and filter, dry the precipitate, crush and sieve it to obtain defatted krill powder; Step S2, sterilization treatment: Add the defatted krill powder to deionized water, adjust the pH to 8.2 - 8.6, sterilize, and cool to room temperature to obtain a sterilized krill culture medium, where the mass ratio of the defatted krill powder to deionized water is 1:76 - 82; Step S3, fermentation: Add an activated composite seed bacterium solution and a fermentation synergist to the sterilized krill culture medium, stir evenly, ferment, centrifuge, take the supernatant, filter by suction, and homogenize to obtain a fermentation suspension, where the mass ratio of the sterilized krill culture medium, the activated composite seed bacterium solution, and the fermentation synergist is 6:0.56 - 1.04:0.16 - 0.2; Step S4, enzymatic hydrolysis: Add a compound enzyme and an enzyme promoter to the fermentation suspension, carry out enzymatic hydrolysis to obtain an enzymatically hydrolyzed fermentation extract, where the mass ratio of the fermentation suspension, the compound enzyme, and the enzyme promoter is 10:0.24 - 0.26:0.06 - 0.14; Step S5, enzyme inactivation: Inactivate the enzymolysis fermentation extract, cool it, centrifuge it, ultrafilter it, and freeze-dry it to obtain Antarctic krill peptide.

[0006] Preferably, in step S1, the mass fraction of the ethanol aqueous solution is 95%.

[0007] Preferably, in step S2, the sterilization temperature is 121 °C and the sterilization time is 15 - 25 min.

[0008] Preferably, in step S3, the fermentation temperature is 28 - 32 °C, the fermentation time is 24 - 36 h. During the homogenization process, control the homogenization pressure to be 100 - 140 MPa, the homogenization temperature to be 72 - 80 °C, and the number of homogenization times to be 6 - 10 times.

[0009] Preferably, in step S4, the enzymolysis temperature is 45 - 55 °C and the enzymolysis time is 5 - 7 h.

[0010] Preferably, in step S5, the enzyme inactivation temperature is 95 - 105 °C and the enzyme inactivation time is 15 - 25 min.

[0011] Preferably, in step S4, the compound enzyme is composed of papain, alkaline protease, and compound protease mixed in a mass ratio of 4:2 - 3:1.

[0012] Preferably, in step S4, the enzyme promoter is composed of calcium gluconate, N-butylpyridine dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and absolute ethanol mixed in a mass ratio of 20:80 - 100:5:7 - 8:1000 - 1200.

[0013] Preferably, in step S1, the activated compound seed bacterium solution is prepared by the following steps: Step S11: Using MRS broth medium as the basic medium, supplement 2 - 3% of carbon source, 0.01 - 0.03% of magnesium sulfate, and 0.002 - 0.004% of manganese sulfate in the basic medium by mass percentage, and adjust the pH value of the system to 5.2 - 5.6 to obtain the activated medium; Step S12: Inoculate the compound bacteria into the activated medium obtained in step S11 at an inoculation amount of 6 - 8% by volume percentage, set the culture temperature to 25 - 30 °C, shake and culture on a shaker, add inorganic calcium source at the 4th h and 6th h of fermentation, the shaker speed is 100 - 200 rpm, and the culture time is 12 - 24 h to obtain the activated compound seed bacterium solution.

[0014] Preferably, in step S12, the compound bacteria are obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a viable bacteria number ratio of 1:0.3 - 0.5 in a sterile environment.

[0015] The Lactobacillus plantarum F-B18-1 is the Lactobacillus plantarum F-B18-1 in Chinese invention patent CN114081854B.

[0016] Preferably, in the step S3, the fermentation synergist is prepared by the following steps: Step A1: Add hydroxyethyl chitosan to an acetic acid aqueous solution, stir until uniform, then add sodium selenite, heat up to 45 - 55 °C, stir and react for 2.2 - 2.8 h, filter, precipitate with alcohol, filter, wash, dry, and grind to obtain modified chitosan, wherein the mass ratio of hydroxyethyl chitosan, acetic acid aqueous solution, and sodium selenite is 15 - 25:120 - 160:1 - 2; Step A2: Ultrasonically disperse the modified chitosan in an acetic acid aqueous solution, adjust the pH of the modified chitosan dispersion, filter, add a surfactant to the filtrate, stir evenly, then add fulvic acid, triacontanol, nicotinic acid, and brewer's yeast powder and continue to stir for 0.4 - 0.6 h to obtain an emulsion; mix the aqueous solution of sodium tripolyphosphate with the emulsion evenly, carry out cross-linking reaction for 0.35 - 0.55 h, after the reaction ends, centrifuge, wash, then disperse the centrifuged product in distilled water and perform ultrasonic treatment, and freeze-dry to obtain the fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, surfactant, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder, and aqueous solution of sodium tripolyphosphate is 1:12 - 18:0.02 - 0.04:0.16 - 0.30:0.6 - 1:0.4 - 0.8:1.2 - 1.6:450 - 550.

[0017] Preferably, in the step A1, the mass fraction of the acetic acid aqueous solution is 1 - 2%.

[0018] Preferably, in the step A2, the mass fraction of the acetic acid aqueous solution is 3 - 5%.

[0019] Preferably, in the step A2, the surfactant is Tween-80 with a mass fraction of 0.06 - 0.08%.

[0020] Preferably, in the step A2, the mass fraction of the aqueous solution of sodium tripolyphosphate is 0.3 - 0.5%.

[0021] In the second aspect, the present invention provides an Antarctic krill peptide prepared by the above method.

[0022] In the third aspect, the present invention provides an application of the Antarctic krill peptide prepared by the above method in skin care products. The present invention has the following beneficial effects: In order to shorten the fermentation and extraction cycle of Antarctic krill peptides and at the same time reduce the average molecular weight of the extracted Antarctic krill peptides, the first aspect of the present invention is to add a fermentation synergist, which contains various active components such as modified chitosan, fulvic acid, triacontanol, nicotinic acid and brewer's yeast powder. The presence of modified chitosan can not only optimize the ionic balance of the fermentation system by chelating metal ions, enhance protease activity, reduce the fermentation cycle, but also serve as a source of organic selenium, and the selenium ions on it can enhance the antioxidant enzyme activity of the bacteria, reduce oxidative stress, improve the activity of the bacteria, and further reduce the fermentation cycle. Fulvic acid can not only combine with calcium and magnesium ions in krill to reduce their inhibitory effect on protease and improve the hydrolysis efficiency, but also contains various active functional groups on it, which can stimulate the enzyme activity of the composite bacteria and further reduce the fermentation cycle. Triacontanol can promote the absorption of nutrients by the bacteria and the secretion of metabolites, improve the activity of the composite bacteria. Nicotinic acid can participate in the catalytic reaction of protease as a coenzyme to improve the substrate conversion efficiency. Brewer's yeast powder can not only serve as a supplementary nitrogen source, but also promote the synthesis of bacterial protein and the secretion of protease. Through the synergistic effect of the above components, the activity of the composite bacteria is enhanced together, and the fermentation cycle is reduced. Second, an enzyme promoter is added. The enzyme promoter contains calcium gluconate. As a calcium ion chelator, calcium gluconate can stabilize the structure of the compound enzyme. N-butylpyridinium dihydrogen phosphate can not only form hydrogen bonds or ionic interactions with polar components (such as polysaccharides and proteins) and calcium gluconate in the fermentation suspension to improve the extraction efficiency and reduce the fermentation cycle, but also the phosphate ions generated by its hydrolysis can enhance the interaction between the compound enzyme and the fermentation suspension, improve the catalytic efficiency of the compound enzyme, and further reduce the average molecular weight of Antarctic krill peptide molecules. Third, a composite bacteria is added. The composite bacteria are prepared by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum in a specific ratio. The acidic environment of Lactobacillus plantarum in the composite bacteria activates the alkaline protease of Brevibacterium flavum to form a complementary pH-adaptive enzyme system. The short-chain fatty acids produced by Lactobacillus plantarum can enhance the colonization ability of Brevibacterium flavum, while the nucleoside degradation products of Brevibacterium flavum provide a carbon source for Lactobacillus plantarum to maintain the metabolic balance of the fermentation system. Through their synergistic effect, the fermentation cycle and the average molecular weight of Antarctic krill peptide molecules are further reduced. Detailed implementation mode

[0023] To make the present invention easier to understand, the following specific examples are used to illustrate the present invention in detail. These examples are only illustrative and are not limited to the application scope of the present invention.

[0024] For those not specifying specific technologies or conditions in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field, or in accordance with the product instructions. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0025] The Antarctic krill powder was purchased from China National Fisheries Corporation. After being transported to the laboratory, it was stored frozen at -20°C. Lactobacillus plantarum F-B18-1 is the Lactobacillus plantarum F-B18-1 in Chinese invention patent CN114081854B. Lactobacillus plantarum ATCC8014 was purchased from Hangzhou Baosai Biotechnology Co., Ltd. Brevibacterium flavum CICC 22619 was purchased from the China Center for Industrial Culture Collection. After being transported to the laboratory and activated, it was stored using glycerol at -70°C. Papain, alkaline protease, and compound protease were all purchased from Shandong Yaotu Biotechnology Co., Ltd.

[0026] Preparation Examples 1-3 and Comparative Preparation Examples 1-2 provide a fermentation synergist.

[0027] Preparation Example 1 This preparation example provides a fermentation synergist, which is prepared by the following steps: Step A1: Add hydroxymethyl chitosan to an acetic acid aqueous solution with a mass fraction of 1%, stir at a rotation speed of 600 rpm for 12 min until homogeneous, then add sodium selenite, raise the temperature to 45°C, maintain the rotation speed unchanged, stir and react for 2.2 h, filter, add ethanol with a volume fraction of 70% for precipitation, filter, wash the filter residue with absolute ethanol, dry to constant weight at 45°C, grind, and pass through a 200-mesh sieve to obtain modified chitosan. Among them, the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, and sodium selenite is 15:120:1; Step A2: Ultrasonically disperse the modified chitosan in an acetic acid aqueous solution with a mass fraction of 3%. Under a ultrasonic frequency of 35 kHz and a ultrasonic power of 500 w, ultrasonicate for 12 min, add a 0.1 M sodium hydroxide aqueous solution to adjust the pH to 4.6, and filter to obtain a filtrate; add Tween-80 with a mass fraction of 0.06%, and stir at a rotation speed of 250 rpm at 25°C for 45 min. Then add fulvic acid, triacontanol, nicotinic acid, and beer yeast powder and stir at a rotation speed of 600 rpm at 25°C for 0.4 h to obtain an emulsion; stir the aqueous solution of tripolyphosphate with a mass fraction of 0.3% and the emulsion at a rotation speed of 600 rpm for 14 min until homogeneous, maintain the rotation speed unchanged, and continue the cross-linking reaction for 0.35 h. After the reaction is completed, centrifuge, wash the centrifuged product 3 times with an aqueous solution of Tween 80 with a mass fraction of 0.06%, then disperse the centrifuged product in distilled water and ultrasonicate at a power of 60 w for 10 min. The freeze-drying temperature is -30°C, the vacuum degree is 12 Pa, and the drying time is 26 h to obtain the fermentation synergist. Among them, the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, beer yeast powder, and aqueous solution of tripolyphosphate is 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0028] Preparation Example 2 This preparation example provides a fermentation synergist, which is prepared by the following steps: Step A1: Add hydroxymethyl chitosan into an acetic acid aqueous solution with a mass fraction of 1.5%, stir at a rotation speed of 650 rpm for 16 min until homogeneous, then add sodium selenite, raise the temperature to 50 °C, stir and react for 2.5 h, filter, add ethanol with a volume fraction of 70% for precipitation, filter, wash the filter residue with absolute ethanol, dry at 55 °C to constant weight, grind, and pass through a 240-mesh sieve to obtain modified chitosan. Among them, the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, and sodium selenite is 20:140:1.5; Step A2: Ultrasonically disperse the modified chitosan in an acetic acid aqueous solution with a mass fraction of 3%, control the ultrasonic frequency at 40 kHz, ultrasonic power at 550 w, ultrasonicate for 16 min, add 0.15 M sodium hydroxide aqueous solution to adjust the pH to 5.0, and filter to obtain a filtrate; add Tween-80 with a mass fraction of 0.07%, and stir at a rotation speed of 300 rpm at 25 °C for 50 min, then add fulvic acid, triacontanol, nicotinic acid, and beer yeast powder and continue to stir at 650 rpm at 25 °C for 0.5 h to obtain an emulsion; stir the aqueous solution of tripolyphosphate with a mass fraction of 0.4% and the emulsion at a rotation speed of 650 rpm for 16 min until homogeneous, maintain the rotation speed unchanged, and continue the cross-linking reaction for 0.45 h. After the reaction is completed, centrifuge, wash the centrifuged product 4 times with an aqueous solution of Tween 80 with a mass fraction of 0.07%, then disperse the centrifuged product in distilled water and ultrasonicate at a power of 70 w for 14 min. The freeze-drying temperature is -25 °C, the vacuum degree is 13 Pa, and the drying time is 28 h to obtain the fermentation synergist. Among them, the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, beer yeast powder, and aqueous solution of tripolyphosphate is 1:15:0.03:0.23:0.8:0.6:1.4:500.

[0029] Preparation Example 3 This preparation example provides a fermentation synergist, which is prepared by the following steps: Step A1: Add hydroxymethyl chitosan into an acetic acid aqueous solution with a mass fraction of 2%, stir at 700 rpm for 20 min until homogeneous, then add sodium selenite, raise the temperature to 55 °C, stir and react for 2.8 h, filter, add ethanol with a volume fraction of 70% for precipitation, filter, wash the filter residue with absolute ethanol, dry at 55 °C to constant weight, grind, and pass through a 280-mesh sieve to obtain modified chitosan. Among them, the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, and sodium selenite is 25:160:2; Step A2: Ultrasonically disperse the modified chitosan in an acetic acid aqueous solution with a mass fraction of 5%. While controlling the ultrasonic frequency at 45 kHz and the ultrasonic power at 600 W, perform ultrasonic treatment for 20 min. Add a 0.2 M sodium hydroxide aqueous solution to adjust the pH to 5.4, and filter to obtain a filtrate. Add Tween-80 with a mass fraction of 0.08%, and stir at a speed of 350 rpm at 25°C for 55 min. Then add fulvic acid, triacontanol, nicotinic acid, and beer yeast powder, and continue to stir at 650 rpm at 25°C for 0.6 h to obtain an emulsion. Mix the aqueous solution of tripolyphosphate with a mass fraction of 0.5% and the emulsion, and stir at a speed of 700 rpm for 18 min until uniform. While maintaining the speed unchanged, continue the cross-linking reaction for 0.55 h. After the reaction ends, centrifuge, wash the centrifuged product 5 times with an aqueous solution of Tween 80 with a mass fraction of 0.08%. Then disperse the centrifuged product in distilled water and perform ultrasonic treatment at a power of 80 W for 18 min. The freeze-drying temperature is -20°C, the vacuum degree is 13 Pa, and the drying time is 30 h to obtain a fermentation synergist. Among them, the mass ratio of the modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, beer yeast powder, and tripolyphosphate aqueous solution is 1:18:0.04:0.30:1:0.8:1.6:550.

[0030] Comparative Preparation Example 1 This comparative preparation example provides a fermentation synergist, which is prepared by the following steps: Step A1: Add hydroxymethyl chitosan to an acetic acid aqueous solution with a mass fraction of 1%. While controlling the speed at 600 rpm, stir for 12 min until uniform. Then add sodium phosphite, heat up to 45°C, while maintaining the speed unchanged, stir and react for 2.2 h, filter, add ethanol with a volume fraction of 70% for precipitation, filter, wash the filter residue with absolute ethanol, dry to constant weight at 45°C, grind, and pass through a 200-mesh sieve to obtain modified chitosan. Among them, the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, and sodium phosphite is 15:120:1; Step A2: Ultrasonically disperse the modified chitosan in an aqueous acetic acid solution with a mass fraction of 3%. Control the ultrasonic frequency at 35 kHz and the ultrasonic power at 500 W, and ultrasonicate for 12 min. Add 0.1 M aqueous sodium hydroxide solution to adjust the pH to 4.6, and filter to obtain a filtrate. Add Tween-80 with a mass fraction of 0.06%, and stir at a speed of 250 rpm at 25 °C for 45 min. Then add fulvic acid, triacontanol, nicotinic acid, and beer yeast powder, and stir at a speed of 600 rpm at 25 °C for 0.4 h to obtain an emulsion. Mix the aqueous solution of tripolyphosphate with a mass fraction of 0.3% and the emulsion, and stir at a speed of 600 rpm for 14 min until homogeneous. Keep the speed unchanged and continue the cross-linking reaction for 0.35 h. After the reaction, centrifuge, wash the centrifuged product 3 times with an aqueous solution of Tween 80 with a mass fraction of 0.06%, then disperse the centrifuged product in distilled water and ultrasonicate at a power of 60 W for 10 min. The freeze-drying temperature is -30 °C, the vacuum degree is 12 Pa, and the drying time is 26 h to obtain the fermentation synergist. Among them, the mass ratio of the modified chitosan, aqueous acetic acid solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, beer yeast powder, and aqueous tripolyphosphate solution is 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0031] Comparative Preparation Example 2 This comparative preparation example provides a fermentation synergist, which is prepared by the following steps: Step A1: Add hydroxymethyl chitosan to an aqueous acetic acid solution with a mass fraction of 1%. Control the stirring speed at 600 rpm and stir for 12 min until homogeneous. Then add sodium selenite, heat up to 45 °C, keep the stirring speed unchanged, and stir and react for 2.2 h. Filter, add ethanol with a volume fraction of 70% for precipitation, filter, wash the filter residue with absolute ethanol, dry at 45 °C to constant weight, grind, and pass through a 200-mesh sieve to obtain the modified chitosan. Among them, the mass ratio of hydroxymethyl chitosan, aqueous acetic acid solution, and sodium selenite is 15:120:1. Step A2: Ultrasonically disperse the modified chitosan in an aqueous acetic acid solution with a mass fraction of 3%. Control the ultrasonic frequency at 35 kHz and the ultrasonic power at 500 W, and ultrasonicate for 12 min. Add a 0.1 M aqueous sodium hydroxide solution to adjust the pH to 4.6, and filter to obtain a filtrate. Add Tween-80 with a mass fraction of 0.06%, and stir at 250 rpm at 25 °C for 45 min. Then add phytic acid, triacontanol, nicotinic acid, and beer yeast powder, and stir at 600 rpm at 25 °C for 0.4 h to obtain an emulsion. Mix an aqueous solution of tripolyphosphate with a mass fraction of 0.3% and the emulsion at 600 rpm and stir for 14 min until homogeneous. Keep the rotation speed unchanged and continue the cross-linking reaction for 0.35 h. After the reaction, centrifuge, wash the centrifuged product 3 times with an aqueous solution of Tween 80 with a mass fraction of 0.06%, then disperse the centrifuged product in distilled water and ultrasonicate at a power of 60 W for 10 min. The freeze-drying temperature is -30 °C, the vacuum degree is 12 Pa, and the drying time is 26 h to obtain a fermentation synergist. Among them, the mass ratio of the modified chitosan, aqueous acetic acid solution, Tween-80, phytic acid, triacontanol, nicotinic acid, beer yeast powder, and aqueous tripolyphosphate solution is 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0032] Preparation Examples 4-6 and Comparative Preparation Examples 3-5 provide an activated composite seed bacterium solution.

[0033] Preparation Example 4 This preparation example provides an activated composite seed bacterium solution, which is prepared by the following steps: Step S11: Use MRS broth medium as the basal medium. By mass percentage, supplement 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate in the basal medium, and use a 0.05 M aqueous ammonium carbonate solution to adjust the pH value of the system to 5.2 to obtain an activated medium. Step S12: Inoculate the composite bacteria into the activated medium obtained in Step S11 at an inoculation amount of 6% by volume. Set the culture temperature at 25 °C and culture with a shaker. Add eggshell powder at the 4th h and 6th h of fermentation, the shaker speed is 100 rpm, and the culture time is 12 h to obtain an activated composite seed bacterium solution. Among them, the composite bacteria are obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a viable bacteria ratio of 1:0.3 in a sterile environment.

[0034] Preparation Example 5 This preparation example provides an activated composite seed bacterium solution, which is prepared by the following steps: Step S11: Using MRS broth medium as the basal medium, add 2.5% sucrose, 0.02% magnesium sulfate, and 0.003% manganese sulfate by mass percentage. Adjust the pH value of the system to 5.4 with 0.06M ammonium carbonate aqueous solution to obtain the activation medium. Step S12: Inoculate the composite strain into the activation medium obtained in Step S11 at an inoculation amount of 7% by volume. Set the culture temperature at 27°C and culture with shaking. Add eggshell powder at the 4th hour and 6th hour of fermentation. The shaking rate is 150 rpm and the culture time is 18 h to obtain the activated composite seed bacterium solution. The composite strain is obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a viable bacteria ratio of 1:0.4 in a sterile environment.

[0035] Preparation Example 6 This preparation example provides an activated composite seed bacterium solution, which is prepared by the following steps: Step S11: Using MRS broth medium as the basal medium, add 3% glucose, 0.03% magnesium sulfate, and 0.004% manganese sulfate by mass percentage. Adjust the pH value of the system to 5.6 with 0.07M ammonium carbonate aqueous solution to obtain the activation medium. Step S12: Inoculate the composite strain into the activation medium obtained in Step S11 at an inoculation amount of 8% by volume. Set the culture temperature at 30°C and culture with shaking. Add eggshell powder at the 4th hour and 6th hour of fermentation. The shaking rate is 200 rpm and the culture time is 24 h to obtain the activated composite seed bacterium solution. The composite strain is obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a viable bacteria ratio of 1:0.5 in a sterile environment.

[0036] Comparative Preparation Example 3 This comparative preparation example provides an activated composite seed bacterium solution, which is prepared by the following steps: Step S11: Using MRS broth medium as the basal medium, add 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate by mass percentage. Adjust the pH value of the system to 5.2 with 0.05M ammonium carbonate aqueous solution to obtain the activation medium. Step S12: Inoculate the composite strain into the activation medium obtained in Step S11 at an inoculation amount of 6% by volume. Set the culture temperature at 25°C and culture with shaking. Add eggshell powder at the 4th hour and 6th hour of fermentation. The shaking rate is 100 rpm and the culture time is 12 h to obtain the activated composite seed bacterium solution. The composite strain is obtained by mixing Lactobacillus plantarum ATCC 8014 and Brevibacterium flavum with a viable bacteria ratio of 1:0.3 in a sterile environment.

[0037] Comparative Preparation Example 4 This comparative preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps: Step S11: Using MRS broth medium as the basal medium, by mass percentage, add 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate to the basal medium, and adjust the pH value of the system to 5.2 with 0.05M ammonium carbonate aqueous solution to obtain the activated medium; Step S12: Inoculate Lactobacillus plantarum F-B18-1 into the activated medium obtained in Step S11 at an inoculation amount of 6% by volume, set the culture temperature at 25°C, shake and culture on a shaker, add eggshell powder at the 4th hour and 6th hour of fermentation, the shaker speed is 100 rpm, and the culture time is 12 hours to obtain the activated composite seed bacterial solution.

[0038] Comparative Preparation Example 5 This comparative preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps: Step S11: Using MRS broth medium as the basal medium, by mass percentage, add 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate to the basal medium, and adjust the pH value of the system to 5.2 with 0.05M ammonium carbonate aqueous solution to obtain the activated medium; Step S12: Inoculate Brevibacterium flavum into the activated medium obtained in Step S11 at an inoculation amount of 6% by volume, set the culture temperature at 25°C, shake and culture on a shaker, add eggshell powder at the 4th hour and 6th hour of fermentation, the shaker speed is 100 rpm, and the culture time is 12 hours to obtain the activated composite seed bacterial solution.

[0039] Examples 1-3 and Comparative Examples 1-6 provide a method for preparing Antarctic krill peptides using Lactobacillus plantarum.

[0040] Example 1 This example provides a method for preparing Antarctic krill peptides using Lactobacillus plantarum, which specifically includes the following steps: Step S1: Degreasing treatment: Add Antarctic krill powder to 95% ethanol aqueous solution according to a material-liquid ratio of 1:12, stir at a speed of 420 rpm for 6.2 h, let it stand and filter, dry the precipitate at 50°C, crush it through a 220-mesh sieve to obtain degreased krill powder; Step S2: Sterilization treatment: Add the degreased krill powder to deionized water, adjust the pH to 8.2 with 0.6M sodium bicarbonate aqueous solution, raise the temperature to 121°C, sterilize for 15 min, and cool to room temperature to obtain the sterilized krill medium, where the mass ratio of the degreased krill powder to deionized water is 1:76; Step S3, Fermentation: Add the activated composite seed bacterial solution prepared in Preparation Example 4 and the fermentation synergist prepared in Preparation Example 1 to the sterilized krill culture medium, stir at a rotation speed of 550 rpm for 16 min until uniform, ferment at 28 °C for 24 h, then centrifuge at 8000 rpm for 10 min, take the supernatant, filter by suction, and then add it to a high-pressure homogenizer. Control the homogenization pressure at 100 MPa and the homogenization temperature at 72 °C, and perform homogenization treatment 6 times to obtain a fermentation suspension. Among them, the mass ratio of the sterilized krill culture medium, the activated composite seed bacterial solution, and the fermentation synergist is 6:0.56:0.16; Step S4, Enzymolysis: Add a compound enzyme and an enzyme promoter to the fermentation suspension, control the temperature at 45 °C, and enzymolyze for 5 h to obtain an enzymolyzed fermentation extract. Among them, the mass ratio of the fermentation suspension, the compound enzyme, and the enzyme promoter is 10:0.24:0.06. The compound enzyme is composed of papain, alkaline protease, and compound protease mixed in a mass ratio of 4:2:1, and the enzyme promoter is composed of calcium gluconate, N-butylpyridinium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and absolute ethanol mixed in a mass ratio of 20:80:5:7:1000; Step S5, Enzyme Inactivation: Inactivate the enzymolyzed fermentation extract at 95 °C for 15 min, cool it to room temperature in an ice-water bath, then centrifuge at 8000 g and 4 °C for 16 min. Transfer the supernatant obtained after centrifugation to a 10 kDa ultrafiltration tube, and centrifuge at 4500 g and 4 °C for 45 min for ultrafiltration. The freeze-drying temperature is -40 °C, the vacuum degree is 11 Pa, and the drying time is 32 h to obtain Antarctic krill peptide.

[0041] Example 2 This example provides a method for preparing Antarctic krill peptide by Lactobacillus plantarum, which specifically includes the following steps: Step S1, Degreasing Treatment: Add Antarctic krill powder to an ethanol aqueous solution with a mass fraction of 95% according to a solid-liquid ratio of 1:14, stir at a rotation speed of 460 rpm for 7.3 h, let it stand and filter, dry the precipitate at 55 °C, pulverize it and pass it through a 240-mesh sieve to obtain degreased krill powder; Step S2, Sterilization Treatment: Add the degreased krill powder to deionized water, adjust the pH to 8.4 with a 0.64 M sodium bicarbonate aqueous solution, raise the temperature to 121 °C, sterilize for 20 min, and cool to room temperature to obtain a sterilized krill culture medium. Among them, the mass ratio of the degreased krill powder and deionized water is 1:79; Step S3, Fermentation: Add the activated composite seed bacterium solution prepared in Preparation Example 5 and the fermentation synergist prepared in Preparation Example 2 to the sterilized krill culture medium, stir at a rotation speed of 580 rpm for 18 min until uniform, ferment at 30 °C for 30 h, then centrifuge at 9000 rpm for 12 min, take the supernatant, filter by suction, and then add it to a high-pressure homogenizer. Control the homogenization pressure to be 120 MPa and the homogenization temperature to be 76 °C, and perform homogenization treatment 8 times to obtain a fermentation suspension. Among them, the mass ratio of the sterilized krill culture medium, the activated composite seed bacterium solution, and the fermentation synergist is 6:0.8:0.18; Step S4, Enzymolysis: Add the compound enzyme and the enzyme promoter to the fermentation suspension, control the temperature to be 50 °C, and enzymolyze for 6 h to obtain an enzymolyzed fermentation extract. Among them, the mass ratio of the fermentation suspension, the compound enzyme, and the enzyme promoter is 10:0.24:0.06. The compound enzyme is composed of papain, alkaline protease, and compound protease mixed in a mass ratio of 4:2.5:1. The enzyme promoter is composed of calcium gluconate, N-butylpyridinium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and absolute ethanol mixed in a mass ratio of 20:90:5:7.5:1100; Step S5, Enzyme Inactivation: Inactivate the enzymolyzed fermentation extract at 100 °C for 20 min, cool it to room temperature in an ice-water bath, then centrifuge at 8500 g and 4 °C for 18 min. Transfer the supernatant obtained after centrifugation to a 10 kDa ultrafiltration tube, centrifuge at 5000 g and 4 °C for 50 min. The freeze-drying temperature is -35 °C, the vacuum degree is 13 Pa, and the drying time is 36 h to obtain Antarctic krill peptide.

[0042] Example 3 This example provides a method for preparing Antarctic krill peptide by Lactobacillus plantarum, which specifically includes the following steps: Step S1, Defatting Treatment: Add Antarctic krill powder to an ethanol aqueous solution with a mass fraction of 95% according to a material-liquid ratio of 1:16, stir at a rotation speed of 500 rpm for 8.4 h, let it stand and filter. Place the precipitate in an oven at 60 °C to dry, crush it and pass it through a 260-mesh sieve to obtain defatted krill powder; Step S2, Sterilization Treatment: Add the defatted krill powder to deionized water, adjust the pH to 8.6 with a 0.68 M sodium bicarbonate aqueous solution, raise the temperature to 121 °C, sterilize for 25 min, and cool to room temperature to obtain a sterilized krill culture medium. Among them, the mass ratio of the defatted krill powder and deionized water is 1:82; Step S3, Fermentation: Add the activated composite seed bacterial solution prepared in Preparation Example 6 and the fermentation synergist prepared in Preparation Example 3 to the sterilized krill culture medium, stir at a rotation speed of 610 rpm for 20 min until uniform, ferment at 32 °C for 36 h, then centrifuge at 10000 rpm for 15 min, take the supernatant, filter by suction, and then add it to a high-pressure homogenizer. Control the homogenization pressure at 140 MPa and the homogenization temperature at 80 °C, and perform homogenization treatment 10 times to obtain a fermentation suspension. Among them, the mass ratio of the sterilized krill culture medium, the activated composite seed bacterial solution, and the fermentation synergist is 6:1.04:0.2; Step S4, Enzymolysis: Add the compound enzyme and the enzyme promoter to the fermentation suspension, control the temperature at 55 °C, and perform enzymolysis for 7 h to obtain an enzymolyzed fermentation extract. Among them, the mass ratio of the fermentation suspension, the compound enzyme, and the enzyme promoter is 10:0.26:0.14. The compound enzyme is composed of papain, alkaline protease, and compound protease mixed in a mass ratio of 4:3:1, and the enzyme promoter is composed of calcium gluconate, N-butylpyridinium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and absolute ethanol mixed in a mass ratio of 20:100:5:8:1200; Step S5, Enzyme Inactivation: Inactivate the enzymolyzed fermentation extract at 105 °C for 25 min, cool it to room temperature in an ice-water bath, then centrifuge at 9000 g and 4 °C for 20 min. Transfer the supernatant obtained after centrifugation to a 10 kDa ultrafiltration tube, centrifuge at 5500 g and 4 °C for 55 min. The freeze-drying temperature is -30 °C, the vacuum degree is 15 Pa, and the drying time is 40 h to obtain Antarctic krill peptide.

[0043] Comparative Example 1 Comparative Example 1 is the same as Example 1, except that the fermentation synergist in Example 1 is replaced with the fermentation synergist prepared in Comparative Preparation Example 1.

[0044] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that the fermentation synergist in Example 1 is replaced with the fermentation synergist prepared in Comparative Preparation Example 2.

[0045] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that the activated composite seed bacterial solution in Example 1 is replaced with the activated composite seed bacterial solution prepared in Comparative Preparation Example 3.

[0046] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that the activated composite seed bacterial solution in Example 1 is replaced with the activated composite seed bacterial solution prepared in Comparative Preparation Example 4.

[0047] Comparative Example 5 Comparative Example 5 was the same as Example 1, except that the activated composite seed bacterial solution in Example 1 was replaced with the activated composite seed bacterial solution prepared in Comparative Preparation Example 5.

[0048] Comparative Example 6 Comparative Example 6 was the same as Example 1, except that the enzyme promoter in Comparative Example 6 was prepared by mixing calcium gluconate, N-butylpyridinium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate and absolute ethanol in a mass ratio of 20:80:5:7:1000.

[0049] Performance Detection 1. Determination of the Yield of Antarctic Krill Peptide Take 0.6 mL of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6 respectively, add 0.6 mL of trichloroacetic acid solution (100 g / L), mix well and let stand for 10 min, then centrifuge at 6000 r / min for 15 min. Take 1 mL of the supernatant into a test tube, add 4 mL of biuret reagent, mix well and let stand for 60 min, and measure the absorbance at 540 nm. Use 1 mL of deionized water instead of the sample as a reference, and bovine serum albumin as a standard product to measure the standard curve. Calculate the mass concentration of Antarctic krill peptide from the standard curve; the calculation of the yield of Antarctic krill peptide is as follows:

[0050] In the formula: c is the mass concentration of Antarctic krill peptide in the fermentation broth, mg / mL; v is the volume of the fermentation broth, mL; m is the mass of defatted shrimp powder, g.

[0051] 2. Molecular Weight Determination The molecular weight was determined by high performance gel permeation chromatography. The chromatographic conditions were as follows: the chromatographic column was SK-GEL G2000SWXL (30.0 cm × 7.8 mm, 5 µm); the column temperature was 30 °C; the detector was a refractive index detector (RID); the mobile phase was an acetonitrile-water-trifluoroacetic acid system (15:85:0.07, V / V / V); the flow rate was 0.6 mL / min. Using human angiotensin II as a standard product (Sigma), calculate the molecular weight of the sample; the specific test data are shown in Table 1.

[0052] Table 1 Performance Tests of Antarctic Krill Peptides Prepared in Examples 1-3 and Comparative Examples 1-6

[0053] As can be seen from Table 1, compared with Comparative Examples 1-6, the Antarctic krill peptides prepared by the methods provided in Examples 1-3 have a higher yield and a smaller average molecular weight.

[0054] 3. Irritation Using the Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6 of the present invention as test samples, the irritation of the above-mentioned Antarctic krill peptides was verified by patch tests. Specifically: 40 volunteers aged 18-50 years were selected. The wells of the patch test device were numbered, and the test samples were respectively placed into the wells with corresponding numbers, with a dosage of 0.02 mL per well. The patch test device with the test sample was applied to the skin of the volunteer's arm with a low-allergy tape, and gently pressed with the palm to make it evenly adhere to the skin. After 24 hours, the patch test device was removed, and the skin condition of the wound area was observed. The identification criteria are shown in Table 2 below.

[0055] Table 2 Irritation test of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6

[0056] The results of the patch test were all negative reactions, that is, the Antarctic krill peptides prepared by the present invention meet the requirements of non-irritation.

[0057] 4. Moisture retention and water locking 80 volunteers aged 18-50 years were selected, with an equal number of men and women, in good health and without skin diseases. They were randomly divided into 10 groups of 10 people each. The Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6 were used on the face respectively. The specific usage method is as follows: After the test volunteers cleaned their facial skin every day, dried it, and evenly applied 2 g of Antarctic krill peptide on the facial skin with their hands.

[0058] The method for testing the skin moisture content is as follows: In an environment with a temperature of 25 °C and a relative humidity of 50%, two 2 cm × 2 cm square experimental areas were drawn at the same position on the left face of the test volunteers. Using a skin moisture meter (provided by CK Company, Germany, model TM300), the facial skin moisture content before using the Antarctic krill peptide and 8 h and 24 h after using it was measured respectively. The measurement results were recorded and their average values were taken. By comparing the test results of each group and through statistical analysis methods, P < 0.05 was obtained, indicating that the difference in the sample means was statistically significant. The specific results are shown in Table 3 below.

[0059] Table 3 Moisture retention and water locking test of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6

[0060] It can be seen from Table 3 that the moisture retention and water locking effects of the Antarctic krill peptides prepared in Examples 1-3 are significantly better than those in Comparative Examples 1-6, and have broad application prospects in the field of cosmetics.

Claims

1. A method for preparing Antarctic krill peptides by Lactobacillus plantarum, characterized in that, It includes the following steps: Step S1, degreasing treatment: Add Antarctic krill powder to the ethanol aqueous solution according to the material-liquid ratio of 1:12 - 16, stir for 6.2 - 8.4 h, let it stand for filtration, dry the precipitate, crush and sieve it to obtain degreased krill powder; Step S2, sterilization treatment: Add the degreased krill powder to deionized water, adjust the pH to 8.2 - 8.6, sterilize it, and cool it to room temperature to obtain a sterilized krill culture medium; Step S3, fermentation: Add the activated composite seed bacteria liquid and a fermentation synergist to the sterilized krill culture medium, stir evenly, ferment, centrifuge, take the supernatant, filter by suction, and homogenize to obtain a fermentation suspension; Step S4, enzymatic hydrolysis: Add a compound enzyme and an enzyme promoter to the fermentation suspension for enzymatic hydrolysis to obtain an enzymatically hydrolyzed fermentation extract; Step S5, enzyme inactivation: Inactivate the enzymatically hydrolyzed fermentation extract, cool it, centrifuge, ultrafilter, and freeze-dry it to obtain Antarctic krill peptides.

2. The method for preparing Antarctic krill peptides by Lactobacillus plantarum according to claim 1, characterized in that, In the said step S3, the mass ratio of the sterilized krill culture medium, the activated composite seed bacteria liquid, and the fermentation synergist is 6:0.56 - 1.04:0.16 - 0.

2.

3. A method for preparing Antarctic krill peptides using Lactobacillus plantarum according to claim 1, characterized in that, In the said step S4, the mass ratio of the fermentation suspension, the compound enzyme, and the enzyme promoter is 10:0.24 - 0.26:0.06 - 0.

14.

4. A method for preparing Antarctic krill peptides using Lactobacillus plantarum according to claim 1, characterized in that, The said activated composite seed bacteria liquid is prepared by the following steps: Step S11, using MRS broth culture medium as the basic culture medium, supplement 2 - 3% of carbon source, 0.01 - 0.03% of magnesium sulfate, and 0.002 - 0.004% of manganese sulfate in the basic culture medium according to the mass percentage, and adjust the pH value of the system to 5.2 - 5.6 to obtain an activated culture medium; Step S12, inoculate the composite bacteria into the activated culture medium obtained in step S11 according to the inoculation amount of 6 - 8% by volume, set the culture temperature at 25 - 30 °C, shake and culture on a shaker, add an inorganic calcium source at the 4th h and 6th h of fermentation, the shaker speed is 100 - 200 rpm, and the culture time is 12 - 24 h to obtain the activated composite seed bacteria liquid.

5. A method for preparing Antarctic krill peptides using Lactobacillus plantarum according to claim 4, characterized in that, The said composite bacteria are obtained by mixing Lactobacillus plantarum F - B18 - 1 and Brevibacterium flavum with a viable bacteria number ratio of 1:0.3 - 0.5 in a sterile environment.

6. A method for preparing Antarctic krill peptides using Lactobacillus plantarum according to claim 1, characterized in that, The said fermentation synergist is prepared by the following steps: Step A1, add hydroxymethyl chitosan to the acetic acid aqueous solution, stir until uniform, then add sodium selenite, heat up to 45 - 55 °C, stir and react for 2.2 - 2.8 h, filter, precipitate with alcohol, filter, wash, dry, and grind to obtain modified chitosan; Step A2, ultrasonically disperse the modified chitosan in the acetic acid aqueous solution, adjust the pH, filter, add a surfactant to the filtrate, stir evenly, then add fulvic acid, triacontanol, nicotinic acid, and beer yeast powder and continue to stir for 0.4 - 0.6 h to obtain an emulsion; mix the aqueous solution of sodium tripolyphosphate with the emulsion evenly, carry out a cross-linking reaction for 0.35 - 0.55 h, after the reaction ends, centrifuge, wash, then disperse the centrifuged product in distilled water and perform ultrasonic treatment, and freeze-dry to obtain the fermentation synergist.

7. The method for preparing Antarctic krill peptides by Lactobacillus plantarum according to claim 6, characterized in that, In the said step A1, the mass ratio of hydroxymethyl chitosan, the acetic acid aqueous solution, and sodium selenite is 15 - 25:120 - 160:1 - 2.

8. A method for preparing Antarctic krill peptides by Lactobacillus plantarum according to claim 7, characterized in that, In the step A2, the mass ratio of the modified chitosan, acetic acid aqueous solution, surfactant, fulvic acid, triacontanol, nicotinic acid, beer yeast powder and tripolyphosphate aqueous solution is 1: 12-18: 0.02-0.04: 0.16-0.30: 0.6-1: 0.4-0.8: 1.2-1.6: 450-550.

9. Antarctic krill peptide prepared by the method for preparing Antarctic krill peptide by Lactobacillus plantarum according to any one of claims 1-8.

10. Use of the Antarctic krill peptide according to claim 9 in skin care products.

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