Method for preparing antarctic krill peptide by lactobacillus plantarum and application thereof

Abstract

The present application relates to the technical field of microorganism, and specifically discloses a method for preparing Antarctic krill peptide by lactobacillus plantarum and application thereof.The method for preparing Antarctic krill peptide by lactobacillus plantarum comprises the following steps: step S1, defatting treatment, step S2, sterilization treatment, step S3, fermentation, step S4, enzymolysis, and step S5, enzyme inactivation.The present application adds fermentation synergist, enzymatic agent and compound bacterial species, and through the synergistic effect thereof, the prepared Antarctic krill peptide not only has high yield, low average molecular weight, small irritation, strong moisturizing and water-locking performance, and wide application prospect in the field of cosmetics.

Description

Technical Field

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

[0002] Antarctic krill meal is rich in protein, astaxanthin, phospholipid fatty acids, and other ingredients, and is often used for krill oil extraction. After oil extraction, the defatted krill meal contains a large amount of high-quality protein, and the preparation of krill peptides with antioxidant and blood pressure-lowering properties has attracted industry attention.

[0003] Bacillus subtilis is widely used in the fermentation and extraction of Antarctic krill peptides due to its advantages such as high enzyme production and rapid growth. However, this extraction process suffers from problems such as long fermentation time, limited efficiency, and autolysis of the bacteria, which leads to a decrease in enzyme production. This ultimately reduces the yield of Antarctic krill peptides. Furthermore, the Antarctic krill peptides obtained using this method have a wide average molecular weight range and a limited antioxidant effect, which cannot meet the moisturizing and hydration requirements of skin care products. Summary of the Invention

[0004] In order to solve the problems mentioned in the above background technology, the present invention provides a method for preparing Antarctic krill peptides using Lactobacillus plantarum and the application of the method in skin care products.

[0005] In a first aspect, the present invention provides a method for preparing Antarctic krill peptides using Lactobacillus plantarum, which adopts the following technical solution:

[0006] A method for preparing Antarctic krill peptide using Lactobacillus plantarum, comprising the following steps:

[0007] Step S1, defatting treatment: adding Antarctic krill powder to ethanol-water solution at a material-liquid ratio of 1:12-16, stirring for 6.2-8.4 hours, standing and filtering, drying the precipitate, crushing and sieving to obtain defatted krill powder;

[0008] Step S2, sterilization treatment: adding defatted krill powder to deionized water, adjusting the pH to 8.2-8.6, sterilizing, and cooling to room temperature to obtain a sterilized krill culture medium, wherein the mass ratio of defatted krill powder to deionized water is 1:76-82;

[0009] Step S3, fermentation: adding an activated composite seed bacterial solution and a fermentation synergist to a sterilized krill culture medium, stirring uniformly, fermenting, centrifuging, collecting the supernatant, filtering, and homogenizing to obtain a fermentation suspension, wherein the mass ratio of the sterilized krill culture medium, the activated composite seed bacterial solution, and the fermentation synergist is 6:0.56-1.04:0.16-0.2;

[0010] Step S4, enzymatic hydrolysis: adding the compound enzyme and the enzymatic agent to the fermentation suspension, performing enzymatic hydrolysis to obtain an enzymatic fermentation extract, wherein the mass ratio of the fermentation suspension, the compound enzyme, and the enzymatic agent is 10:0.24-0.26:0.06-0.14;

[0011] Step S5, enzyme inactivation: inactivating the enzyme in the enzymatic fermentation extract, cooling, centrifuging, ultrafiltration, and freeze-drying to obtain Antarctic krill peptide.

[0012] Preferably, in step S1, the mass fraction of the ethanol aqueous solution is 95%.

[0013] Preferably, in step S2, the sterilization temperature is 121° C. and the sterilization time is 15-25 minutes.

[0014] Preferably, in step S3, the fermentation temperature is 28-32° C., the fermentation time is 24-36 h, and during the homogenization process, the homogenization pressure is controlled to be 100-140 MPa, the homogenization temperature is 72-80° C., and the homogenization times are 6-10 times.

[0015] Preferably, in step S4, the enzymatic hydrolysis temperature is 45-55° C., and the enzymatic hydrolysis time is 5-7 h.

[0016] Preferably, in step S5, the enzyme inactivation temperature is 95-105° C., and the enzyme inactivation time is 15-25 min.

[0017] Preferably, in step S4, the composite enzyme is prepared by mixing papain, alkaline protease and composite protease in a mass ratio of 4:2-3:1.

[0018] Preferably, in step S4, the enzymatic agent is prepared by mixing calcium gluconate, N-butylpyridinium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate and anhydrous ethanol in a mass ratio of 20:80-100:5:7-8:1000-1200.

[0019] Preferably, in step S1, the activated composite seed bacterial solution is prepared by the following steps:

[0020] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2-3% carbon source, 0.01-0.03% magnesium sulfate, and 0.002-0.004% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2-5.6 to obtain an activated medium;

[0021] Step S12, inoculate the composite strain into the activation culture medium obtained in step S11 at an inoculum size of 6-8% by volume, set the culture temperature to 25-30°C, culture on a shaker, add an inorganic calcium source at the 4th and 6th hours of fermentation, set the shaking rate to 100-200 rpm, and culture for 12-24 hours to obtain an activated composite seed bacterial solution.

[0022] Preferably, in step S12, the composite bacteria is obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum at a live bacteria ratio of 1:0.3-0.5 in a sterile environment.

[0023] The Lactobacillus plantarum F-B18-1 is the Lactobacillus plantarum F-B18-1 in Chinese invention patent CN114081854B.

[0024] Preferably, in step S3, the fermentation synergist is prepared by the following steps:

[0025] Step A1, adding hydroxymethyl chitosan to an acetic acid aqueous solution, stirring until uniform, then adding sodium selenite, heating to 45-55° C., stirring and reacting for 2.2-2.8 hours, filtering, alcohol precipitation, filtering, washing, drying, and grinding to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution and sodium selenite is 15-25:120-160:1-2;

[0026] Step A2: ultrasonically dispersing the modified chitosan in an acetic acid aqueous solution, adjusting the pH of the modified chitosan dispersion, filtering, adding a surfactant to the filtrate, stirring evenly, then adding fulvic acid, triacontanol, nicotinic acid, and brewer's yeast powder and continuing to stir for 0.4-0.6 hours to obtain an emulsion; uniformly mixing the tripolyphosphate aqueous solution and the emulsion, performing a cross-linking reaction for 0.35-0.55 hours, centrifuging and washing after the reaction, and then dispersing the centrifuged product in distilled water, ultrasonically treating it, and freeze-drying it to obtain a fermentation synergist, wherein the mass ratio of the modified chitosan, the acetic acid aqueous solution, the surfactant, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder, and the 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.

[0027] Preferably, in step A1, the mass fraction of the acetic acid aqueous solution is 1-2%.

[0028] Preferably, in step A2, the mass fraction of the acetic acid aqueous solution is 3-5%.

[0029] Preferably, in step A2, the surfactant is Tween-80 with a mass fraction of 0.06-0.08%.

[0030] Preferably, in step A2, the mass fraction of the tripolyphosphate aqueous solution is 0.3-0.5%.

[0031] In a second aspect, the present invention provides an Antarctic krill peptide prepared by the above method.

[0032] In a third aspect, the present invention provides an application of the Antarctic krill peptide prepared by the above method in skin care products.

[0033] The present invention has the following beneficial effects:

[0034] In order to shorten the fermentation and extraction cycle of Antarctic krill peptides and reduce the average molecular weight of the extracted Antarctic krill peptides, the present invention first adds a fermentation synergist, which contains multiple 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 ion balance of the fermentation system by chelating metal ions, enhance protease activity and shorten the fermentation cycle, but also serve as a source of organic selenium. The selenium ions on it can enhance the activity of bacterial antioxidant enzymes, reduce oxidative stress, improve bacterial activity, and further shorten the fermentation cycle. During the fermentation period, fulvic acid can not only combine with calcium and magnesium ions in krill to reduce their inhibitory effect on proteases and improve hydrolysis efficiency, but also contains a variety of active functional groups that can stimulate the enzyme activity of the composite bacteria and further reduce the fermentation cycle. Triacontanol can promote the absorption of nutrients and the secretion of metabolites by the bacteria, thereby improving the activity of the composite bacteria. Nicotinic acid can participate in the catalytic reaction of proteases as a coenzyme and improve the efficiency of substrate conversion. Brewer's yeast powder can not only serve as a supplementary nitrogen source, but also promote bacterial protein synthesis and protease secretion. Through the synergistic effect of the above components, The activity of the composite bacteria is enhanced and the fermentation cycle is shortened. Secondly, an enzymatic agent is added, which contains calcium gluconate. Calcium gluconate, as a calcium ion chelator, can stabilize the structure of the composite enzyme. N-butylpyridinium dihydrogen phosphate can not only form hydrogen bonds or ion interactions with polar components (such as polysaccharides, proteins) and calcium gluconate in the fermentation suspension, thereby improving the extraction efficiency and shortening the fermentation cycle, but also the phosphate ions produced by its hydrolysis can enhance the interaction between the composite enzyme and the fermentation suspension, thereby improving the catalytic efficiency of the composite enzyme and further reducing the Antarctic krill. The average molecular weight of the peptide molecules; thirdly, a composite bacterial strain is added. The composite bacterial strain is made by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum in a specific proportion. The acidic environment of Lactobacillus plantarum in the composite bacterial strain 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, maintaining 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 DESCRIPTION

[0035] In order to make the present invention easier to understand, the present invention is described in detail below with reference to specific embodiments. These embodiments are only for illustration and do not limit the scope of application of the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature or the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] Antarctic krill meal was purchased from China National Fisheries Co., Ltd. and stored frozen at −20°C after delivery to the laboratory. Lactobacillus plantarum F-B18-1 is the same strain listed 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 Industrial Microbiological Culture Collection Center. After delivery to the laboratory and activation, it was stored in glycerol and frozen at −70°C. Papain, alkaline protease, and composite protease were all purchased from Shandong Yaotu Bioengineering Co., Ltd.

[0038] Preparation Examples 1-3 and Comparative Preparation Examples 1-2 provide a fermentation enhancer.

[0039] Preparation Example 1

[0040] This preparation example provides a fermentation synergist, which is prepared by the following steps:

[0041] Step A1, adding hydroxymethyl chitosan to a 1% mass fraction acetic acid aqueous solution, controlling the speed to 600 rpm and stirring for 12 minutes until uniform, then adding sodium selenite, heating to 45°C, maintaining the speed unchanged, stirring and reacting for 2.2 hours, filtering, adding 70% volume fraction ethanol to precipitate, filtering, washing the filter residue with anhydrous ethanol, drying at 45°C to constant weight, grinding, and passing through a 200 mesh sieve to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution and sodium selenite is 15:120:1;

[0042] Step A2, ultrasonically disperse the modified chitosan in a 3% acetic acid aqueous solution, control the ultrasonic frequency to 35kHz, the ultrasonic power to 500w, ultrasonicate for 12min, add 0.1M sodium hydroxide aqueous solution to adjust the pH to 4.6, and filter to obtain a filtrate; add 0.06% Tween-80 by mass, and stir at 250rpm at 25°C for 45min, then add fulvic acid, triacontanol, nicotinic acid and brewer's yeast powder and stir at 600rpm at 25°C for 0.4h to obtain an emulsion; add 0.3% tripolyphosphate aqueous solution and the emulsion and stir at 600rpm for 1min. The mixture was stirred for 4 minutes until uniform, the speed was maintained unchanged, and the cross-linking reaction was continued for 0.35 hours. After the reaction was completed, the mixture was centrifuged and the centrifuged product was washed three times with a 0.06% Tween 80 aqueous solution. The centrifuged product was then dispersed in distilled water and ultrasonically treated at a power of 60W for 10 minutes. The freeze-drying temperature was -30°C, the vacuum degree was 12Pa, and the drying time was 26 hours to obtain a fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder and tripolyphosphate aqueous solution was 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0043] Preparation Example 2

[0044] This preparation example provides a fermentation synergist, which is prepared by the following steps:

[0045] Step A1, adding hydroxymethyl chitosan to a 1.5% mass fraction acetic acid aqueous solution, controlling the speed to 650 rpm and stirring for 16 minutes until uniform, then adding sodium selenite, heating to 50°C, stirring and reacting for 2.5 hours, filtering, adding 70% volume fraction ethanol to precipitate, filtering, washing the filter residue with anhydrous ethanol, drying at 55°C to constant weight, grinding, and passing through a 240 mesh sieve to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution and sodium selenite is 20:140:1.5;

[0046] Step A2, ultrasonically disperse the modified chitosan in a 3% acetic acid aqueous solution, control the ultrasonic frequency to 40kHz, the ultrasonic power to 550w, ultrasonicate for 16min, add 0.15M sodium hydroxide aqueous solution to adjust the pH to 5.0, and filter to obtain a filtrate; add 0.07% Tween-80, and stir at 300rpm at 25°C for 50min, then add fulvic acid, triacontanol, nicotinic acid and brewer's yeast powder and continue stirring at 650rpm at 25°C for 0.5h to obtain an emulsion; add 0.4% tripolyphosphate aqueous solution and the emulsion and stir at 650rpm for 1h The mixture was stirred for 6 minutes until uniform, the speed was maintained unchanged, and the cross-linking reaction was continued for 0.45 hours. After the reaction was completed, the mixture was centrifuged, and the centrifuged product was washed 4 times with a Tween 80 aqueous solution with a mass fraction of 0.07%. The centrifuged product was then dispersed in distilled water and ultrasonically treated at a power of 70 W for 14 minutes. The freeze-drying temperature was -25°C, the vacuum degree was 13 Pa, and the drying time was 28 hours to obtain a fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder and tripolyphosphate aqueous solution was 1:15:0.03:0.23:0.8:0.6:1.4:500.

[0047] Preparation Example 3

[0048] This preparation example provides a fermentation synergist, which is prepared by the following steps:

[0049] Step A1, adding hydroxymethyl chitosan to a 2% by mass acetic acid aqueous solution, stirring at 700 rpm for 20 minutes until uniform, then adding sodium selenite, heating to 55°C, stirring and reacting for 2.8 hours, filtering, adding 70% by volume ethanol to precipitate, filtering, washing the filter residue with anhydrous ethanol, drying at 55°C to constant weight, grinding, and passing through a 280-mesh sieve to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, and sodium selenite is 25:160:2;

[0050] Step A2, ultrasonically disperse the modified chitosan in a 5% acetic acid aqueous solution, control the ultrasonic frequency to 45kHz, the ultrasonic power to 600w, ultrasonicate for 20min, add 0.2M sodium hydroxide aqueous solution to adjust the pH to 5.4, and filter to obtain a filtrate; add 0.08% Tween-80 by mass, and stir at 350rpm at 25°C for 55min, then add fulvic acid, triacontanol, nicotinic acid and brewer's yeast powder and continue stirring at 650rpm at 25°C for 0.6h to obtain an emulsion; stir the emulsion at a speed of 700rpm with a 0.5% tripolyphosphate aqueous solution. The mixture was stirred for 18 minutes until uniform, the speed was maintained unchanged, and the cross-linking reaction was continued for 0.55 hours. After the reaction was completed, the mixture was centrifuged, and the centrifuged product was washed 5 times with a Tween 80 aqueous solution with a mass fraction of 0.08%. The centrifuged product was then dispersed in distilled water and ultrasonically treated at a power of 80 W for 18 minutes. The freeze-drying temperature was -20°C, the vacuum degree was 13 Pa, and the drying time was 30 hours to obtain a fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder and tripolyphosphate aqueous solution was 1:18:0.04:0.30:1:0.8:1.6:550.

[0051] Comparative Preparation Example 1

[0052] This comparative preparation example provides a fermentation synergist, which is prepared by the following steps:

[0053] Step A1, adding hydroxymethyl chitosan to a 1% mass fraction acetic acid aqueous solution, controlling the speed to 600 rpm and stirring for 12 minutes until uniform, then adding sodium phosphite, heating to 45°C, maintaining the speed unchanged, stirring and reacting for 2.2 hours, filtering, adding 70% volume fraction ethanol to precipitate, filtering, washing the filter residue with anhydrous ethanol, drying at 45°C to constant weight, grinding, and passing through a 200 mesh sieve to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution and sodium phosphite is 15:120:1;

[0054] Step A2, ultrasonically disperse the modified chitosan in a 3% acetic acid aqueous solution, control the ultrasonic frequency to 35kHz, the ultrasonic power to 500w, ultrasonicate for 12min, add 0.1M sodium hydroxide aqueous solution to adjust the pH to 4.6, and filter to obtain a filtrate; add 0.06% Tween-80 by mass, and stir at 250rpm at 25°C for 45min, then add fulvic acid, triacontanol, nicotinic acid and brewer's yeast powder and stir at 600rpm at 25°C for 0.4h to obtain an emulsion; add 0.3% tripolyphosphate aqueous solution and the emulsion and stir at 600rpm for 1min. The mixture was stirred for 4 minutes until uniform, the speed was maintained unchanged, and the cross-linking reaction was continued for 0.35 hours. After the reaction was completed, the mixture was centrifuged and the centrifuged product was washed three times with a 0.06% Tween 80 aqueous solution. The centrifuged product was then dispersed in distilled water and ultrasonically treated at a power of 60W for 10 minutes. The freeze-drying temperature was -30°C, the vacuum degree was 12Pa, and the drying time was 26 hours to obtain a fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, fulvic acid, triacontanol, nicotinic acid, brewer's yeast powder and tripolyphosphate aqueous solution was 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0055] Comparative Preparation Example 2

[0056] This comparative preparation example provides a fermentation synergist, which is prepared by the following steps:

[0057] Step A1, adding hydroxymethyl chitosan to a 1% mass fraction acetic acid aqueous solution, controlling the speed to 600 rpm and stirring for 12 minutes until uniform, then adding sodium selenite, heating to 45°C, maintaining the speed unchanged, stirring and reacting for 2.2 hours, filtering, adding 70% volume fraction ethanol to precipitate, filtering, washing the filter residue with anhydrous ethanol, drying at 45°C to constant weight, grinding, and passing through a 200 mesh sieve to obtain modified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution and sodium selenite is 15:120:1;

[0058] Step A2, ultrasonically disperse the modified chitosan in a 3% acetic acid aqueous solution, control the ultrasonic frequency to 35kHz, the ultrasonic power to 500w, ultrasonicate for 12min, add 0.1M sodium hydroxide aqueous solution to adjust the pH to 4.6, and filter to obtain a filtrate; add 0.06% Tween-80, and stir at 250rpm at 25°C for 45min, then add phytic acid, triacontanol, nicotinic acid and brewer's yeast powder and stir at 600rpm at 25°C for 0.4h to obtain an emulsion; add 0.3% tripolyphosphate aqueous solution and the emulsion and stir at 600rpm for 1min. The mixture was stirred for 4 minutes until uniform, the speed was maintained unchanged, and the cross-linking reaction was continued for 0.35 hours. After the reaction was completed, the mixture was centrifuged and the centrifuged product was washed three times with a 0.06% mass fraction of Tween 80 aqueous solution. The centrifuged product was then dispersed in distilled water and ultrasonically treated at a power of 60W for 10 minutes. The freeze-drying temperature was -30°C, the vacuum degree was 12Pa, and the drying time was 26 hours to obtain a fermentation synergist, wherein the mass ratio of modified chitosan, acetic acid aqueous solution, Tween-80, phytic acid, triacontanol, nicotinic acid, brewer's yeast powder and tripolyphosphate aqueous solution was 1:12:0.02:0.16:0.6:0.4:1.2:450.

[0059] Preparation Examples 4-6 and Comparative Preparation Examples 3-5 provide an activated composite seed bacterial solution.

[0060] Preparation Example 4

[0061] This preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0062] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2 with a 0.05 M ammonium carbonate aqueous solution to obtain an activated medium;

[0063] Step S12, inoculating the composite bacteria into the activation culture medium obtained in step S11 at an inoculum rate of 6% by volume, setting the culture temperature to 25°C, shaking the culture on a shaker, adding eggshell powder at the 4th and 6th hours of fermentation, the shaking rate being 100 rpm, and the culture time being 12 hours to obtain an activated composite seed bacterial solution, wherein the composite bacteria are obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a live bacterial count ratio of 1:0.3 in a sterile environment.

[0064] Preparation Example 5

[0065] This preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0066] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2.5% sucrose, 0.02% magnesium sulfate, and 0.003% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.4 with a 0.06 M ammonium carbonate aqueous solution to obtain an activated medium;

[0067] Step S12, inoculating the composite strain into the activated culture medium obtained in step S11 at an inoculum rate of 7% by volume, setting the culture temperature to 27°C, shaking the culture on a shaker, adding eggshell powder at the 4th and 6th hours of fermentation, the shaking rate being 150 rpm, and the culture time being 18 hours to obtain an activated composite seed bacterial solution, wherein the composite strain is obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a live bacterial count ratio of 1:0.4 in a sterile environment.

[0068] Preparation Example 6

[0069] This preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0070] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 3% glucose, 0.03% magnesium sulfate, and 0.004% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.6 with a 0.07 M ammonium carbonate aqueous solution to obtain an activated medium;

[0071] Step S12, inoculating the composite bacteria into the activation culture medium obtained in step S11 at an inoculum rate of 8% by volume, setting the culture temperature to 30°C, shaking the culture on a shaker, adding eggshell powder at the 4th and 6th hours of fermentation, the shaking rate being 200 rpm, and the culture time being 24 hours to obtain an activated composite seed bacterial solution, wherein the composite bacteria are obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum with a live bacterial count ratio of 1:0.5 in a sterile environment.

[0072] Comparative Preparation Example 3

[0073] This comparative preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0074] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2 with a 0.05 M ammonium carbonate aqueous solution to obtain an activated medium;

[0075] Step S12, inoculating the composite strain into the activation culture medium obtained in step S11 at an inoculum rate of 6% by volume, setting the culture temperature to 25° C., shaking culture, adding eggshell powder at the 4th and 6th hours of fermentation, the shaking rate is 100 rpm, and the culture time is 12 hours to obtain an activated composite seed bacterial solution, wherein the composite strain is obtained by mixing Lactobacillus plantarum ATCC 8014 and Brevibacterium flavum at a viable cell count ratio of 1:0.3 in a sterile environment.

[0076] Comparative Preparation Example 4

[0077] This comparative preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0078] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2 with a 0.05 M ammonium carbonate aqueous solution to obtain an activated medium;

[0079] Step S12, inoculating Lactobacillus plantarum F-B18-1 into the activation medium obtained in step S11 at an inoculum size of 6% by volume, setting the culture temperature to 25°C, shaking culture on a shaker, adding eggshell powder at the 4th and 6th hours of fermentation, the shaking rate being 100 rpm, and the culture time being 12 hours to obtain an activated composite seed bacterial solution.

[0080] Comparative Preparation Example 5

[0081] This comparative preparation example provides an activated composite seed bacterial solution, which is prepared by the following steps:

[0082] Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2% glucose, 0.01% magnesium sulfate, and 0.002% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2 with a 0.05 M ammonium carbonate aqueous solution to obtain an activated medium;

[0083] Step S12, inoculate the activated culture medium obtained in step S11 with Brevibacterium flavum at an inoculum rate of 6% by volume, set the culture temperature to 25°C, culture on a shaker, add eggshell powder at the 4th and 6th hours of fermentation, set the shaking speed to 100 rpm, and culture for 12 hours to obtain an activated composite seed bacterial solution.

[0084] Examples 1-3 and Comparative Examples 1-6 provide a method for preparing Antarctic krill peptide using Lactobacillus plantarum.

[0085] Example 1

[0086] This embodiment provides a method for preparing Antarctic krill peptide with Lactobacillus plantarum, which specifically comprises the following steps:

[0087] Step S1, defatting treatment: adding Antarctic krill powder to a 95% ethanol aqueous solution at a material-liquid ratio of 1:12, stirring at a speed of 420 rpm for 6.2 hours, standing and filtering, drying the precipitate at 50° C., and pulverizing through a 220-mesh sieve to obtain defatted krill powder;

[0088] Step S2, sterilization treatment: adding defatted krill powder to deionized water, adjusting the pH to 8.2 with a 0.6M sodium bicarbonate aqueous solution, heating to 121°C, sterilizing for 15 minutes, and cooling to room temperature to obtain a sterilized krill culture medium, wherein the mass ratio of defatted krill powder to deionized water is 1:76;

[0089] Step S3, fermentation: adding 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, controlling the speed to 550 rpm and stirring for 16 minutes until uniform, controlling the fermentation at 28° C. for 24 hours, and then centrifuging at 8000 rpm for 10 minutes, taking the supernatant, filtering, and then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 100 MPa and the homogenization temperature to 72° C., and homogenizing 6 times to obtain a fermentation suspension, wherein 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;

[0090] Step S4, enzymatic hydrolysis: adding a composite enzyme and an enzymatic agent to the fermentation suspension, controlling the temperature at 45° C., and performing enzymatic hydrolysis for 5 hours to obtain an enzymatic fermentation extract, wherein the mass ratio of the fermentation suspension, the composite enzyme, and the enzymatic agent is 10:0.24:0.06, the composite enzyme is prepared by mixing papain, alkaline protease, and composite protease in a mass ratio of 4:2:1, and the enzymatic agent is prepared by mixing calcium gluconate, N-butylpyridine dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous ethanol in a mass ratio of 20:80:5:7:1000;

[0091] Step S5, enzyme inactivation: The enzymatic fermentation extract was inactivated at 95°C for 15 minutes, cooled to room temperature in an ice-water bath, and then centrifuged at 8000g and 4°C for 16 minutes. The supernatant obtained after centrifugation was transferred to a 10 kDa ultrafiltration tube, centrifuged at 4500g and 4°C for 45 minutes, ultrafiltered, and freeze-dried at -40°C, a vacuum degree of 11 Pa, and a drying time of 32 hours to obtain Antarctic krill peptide.

[0092] Example 2

[0093] This embodiment provides a method for preparing Antarctic krill peptide with Lactobacillus plantarum, which specifically comprises the following steps:

[0094] Step S1, defatting treatment: adding Antarctic krill powder to a 95% ethanol aqueous solution at a material-liquid ratio of 1:14, stirring at a speed of 460 rpm for 7.3 hours, standing and filtering, drying the precipitate at 55° C., and pulverizing through a 240-mesh sieve to obtain defatted krill powder;

[0095] Step S2, sterilization treatment: adding defatted krill powder to deionized water, adjusting the pH to 8.4 with a 0.64M sodium bicarbonate aqueous solution, heating to 121°C, sterilizing for 20 minutes, and cooling to room temperature to obtain a sterilized krill culture medium, wherein the mass ratio of defatted krill powder to deionized water is 1:79;

[0096] Step S3, fermentation: adding the activated composite seed bacterial solution prepared in Preparation Example 5 and the fermentation synergist prepared in Preparation Example 2 to the sterilized krill culture medium, controlling the speed to 580 rpm and stirring for 18 minutes until uniform, controlling the fermentation at 30° C. for 30 hours, and then centrifuging at 9000 rpm for 12 minutes, taking the supernatant, filtering, and then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 120 MPa and the homogenization temperature to 76° C., and homogenizing 8 times to obtain a fermentation suspension, wherein the mass ratio of the sterilized krill culture medium, the activated composite seed bacterial solution, and the fermentation synergist is 6:0.8:0.18;

[0097] Step S4, enzymatic hydrolysis: adding a composite enzyme and an enzymatic agent to the fermentation suspension, controlling the temperature at 50° C., and performing enzymatic hydrolysis for 6 hours to obtain an enzymatic fermentation extract, wherein the mass ratio of the fermentation suspension, the composite enzyme, and the enzymatic agent is 10:0.24:0.06, the composite enzyme is composed of papain, alkaline protease, and composite protease in a mass ratio of 4:2.5:1, and the enzymatic agent is composed of calcium gluconate, N-butylpyridine dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous ethanol in a mass ratio of 20:90:5:7.5:1100;

[0098] Step S5, enzyme inactivation: the enzymatic fermentation extract was inactivated at 100°C for 20 minutes, cooled to room temperature in an ice-water bath, and then centrifuged at 8500g and 4°C for 18 minutes. The supernatant obtained after centrifugation was transferred to a 10 kDa ultrafiltration tube, centrifuged at 5000g and 4°C for 50 minutes, and freeze-dried at a temperature of -35°C, a vacuum degree of 13 Pa, and a drying time of 36 hours to obtain Antarctic krill peptide.

[0099] Example 3

[0100] This embodiment provides a method for preparing Antarctic krill peptide with Lactobacillus plantarum, which specifically comprises the following steps:

[0101] Step S1, defatting: adding Antarctic krill powder to a 95% ethanol aqueous solution at a material-liquid ratio of 1:16, stirring at a speed of 500 rpm for 8.4 hours, standing and filtering, drying the precipitate at 60° C., and pulverizing through a 260-mesh sieve to obtain defatted krill powder;

[0102] Step S2, sterilization treatment: adding defatted krill powder to deionized water, adjusting the pH to 8.6 with a 0.68M sodium bicarbonate aqueous solution, heating to 121°C, sterilizing for 25 minutes, and cooling to room temperature to obtain a sterilized krill culture medium, wherein the mass ratio of defatted krill powder to deionized water is 1:82;

[0103] Step S3, fermentation: adding 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, controlling the speed to 610 rpm and stirring for 20 minutes until uniform, controlling the fermentation at 32° C. for 36 hours, and then centrifuging at 10,000 rpm for 15 minutes, taking the supernatant, filtering, and then adding it to a high-pressure homogenizer, controlling the homogenization pressure to 140 MPa and the homogenization temperature to 80° C., and homogenizing 10 times to obtain a fermentation suspension, wherein 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;

[0104] Step S4, enzymatic hydrolysis: adding a composite enzyme and an enzymatic agent to the fermentation suspension, controlling the temperature at 55° C., and performing enzymatic hydrolysis for 7 hours to obtain an enzymatic fermentation extract, wherein the mass ratio of the fermentation suspension, the composite enzyme, and the enzymatic agent is 10:0.26:0.14, the composite enzyme is prepared by mixing papain, alkaline protease, and composite protease in a mass ratio of 4:3:1, and the enzymatic agent is prepared by mixing calcium gluconate, N-butylpyridine dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous ethanol in a mass ratio of 20:100:5:8:1200;

[0105] Step S5, enzyme inactivation: The enzymatic fermentation extract was inactivated at 105°C for 25 minutes, cooled to room temperature in an ice-water bath, and then centrifuged at 9000g and 4°C for 20 minutes. The supernatant obtained after centrifugation was transferred to a 10 kDa ultrafiltration tube, centrifuged at 5500g and 4°C for 55 minutes, and freeze-dried at -30°C, a vacuum degree of 15 Pa, and a drying time of 40 hours to obtain Antarctic krill peptide.

[0106] Comparative Example 1

[0107] Comparative Example 1 is the same as Example 1, except that the fermentation synergist in Example 1 is replaced by the fermentation synergist prepared in Comparative Preparation Example 1.

[0108] Comparative Example 2

[0109] Comparative Example 2 is the same as Example 1, except that the fermentation synergist in Example 1 is replaced by the fermentation synergist prepared in Comparative Preparation Example 2.

[0110] Comparative Example 3

[0111] Comparative Example 3 is the same as Example 1, except that the activated composite seed bacterial solution in Example 1 is replaced by the activated composite seed bacterial solution prepared in Comparative Preparation Example 3.

[0112] Comparative Example 4

[0113] Comparative Example 4 is the same as Example 1, except that the activated composite seed bacterial solution in Example 1 is replaced by the activated composite seed bacterial solution prepared in Comparative Preparation Example 4.

[0114] Comparative Example 5

[0115] Comparative Example 5 is the same as Example 1, except that the activated composite seed bacterial solution in Example 1 is replaced by the activated composite seed bacterial solution prepared in Comparative Preparation Example 5.

[0116] Comparative Example 6

[0117] Comparative Example 6 is the same as Example 1, except that the enzymatic agent in Comparative Example 6 is a mixture of calcium gluconate, N-butylpyridinium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate and anhydrous ethanol in a mass ratio of 20:80:5:7:1000.

[0118] Performance testing

[0119] 1. Determination of Antarctic krill peptide yield

[0120] 0.6 mL of the Antarctic krill peptide prepared in Examples 1-3 and Comparative Examples 1-6 was taken, 0.6 mL of trichloroacetic acid solution (100 g / L) was added, mixed and allowed to stand for 10 minutes, centrifuged at 6000 r / min for 15 minutes, 1 mL of the supernatant was taken into a test tube, 4 mL of biuret reagent was added, mixed and allowed to stand for 60 minutes, and the absorbance was measured at 540 nm. 1 mL of deionized water was used instead of the sample as a reference, bovine serum albumin was used as a standard, and a standard curve was determined. The mass concentration of the Antarctic krill peptide was calculated from the standard curve; the calculation of the Antarctic krill peptide yield was:

[0121]

[0122] Where: 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.

[0123] 2. Molecular mass determination

[0124] Molecular weights were determined by high-performance gel exclusion chromatography (HPLC) using the following conditions: SK-GELG2000SWXL column (30.0 cm × 7.8 mm, 5 µm); column temperature 30°C; differential index detection (RID); mobile phase acetonitrile-water-trifluoroacetic acid (15:85:0.07, v / v / v); flow rate 0.6 mL / min. Molecular weights of the samples were calculated using human angiotensin II (Sigma) as a standard. Specific test data are shown in Table 1.

[0125] Table 1 Performance test of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6

[0126]

[0127] It can be seen from Table 1 that, compared with Comparative Examples 1-6, the Antarctic krill peptides prepared by the methods provided in Examples 1-3 have higher yields and smaller average molecular weights.

[0128] 3. Irritation

[0129] The Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6 of the present invention were used as test samples, and a patch test was performed to verify the irritation of the Antarctic krill peptides. Specifically, 40 volunteers aged between 18 and 50 were selected, the wells of the patch tester were numbered, and the test samples were placed in the corresponding numbered wells of the patch tester at a dosage of 0.02 mL per well. The patch tester containing the test sample was applied to the skin of the arm of the test volunteer using hypoallergenic tape, and the patch was evenly applied to the skin with light pressure using the palm of the hand. The patch tester was removed after 24 hours, and the skin condition at the incision site was observed. The identification criteria are shown in Table 2 below.

[0130] Table 2 Irritation test of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6

[0131]

[0132] The patch test results all showed negative reactions, which means that the Antarctic krill peptide prepared by the present invention meets the requirement of being non-irritating.

[0133] 4. Moisturizing and water locking

[0134] 80 volunteers aged between 18 and 50 years old, half male and half female, in good health and without skin diseases were selected and randomly divided into 10 groups, with 10 participants in each group. The Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6 were applied to the face, respectively. The specific application method was as follows: the volunteers cleaned their facial skin every day, wiped it dry, and evenly applied 2 g of Antarctic krill peptide to the facial skin by hand.

[0135] The skin moisture content test method is as follows: In an environment with a temperature of 25°C and a relative humidity of 50%, two 2cm x 2cm square test areas were drawn on the same location on the left cheek of the test volunteer. Using a skin moisture meter (CK, Germany, model TM300), the facial skin moisture content was measured before application of Antarctic krill peptide and 8 and 24 hours after application. The results were recorded and averaged. Comparison of the test results between the groups was statistically significant when P < 0.05 was achieved through statistical analysis. Specific results are shown in Table 3 below.

[0136] Table 3 Moisturizing and water-locking test of Antarctic krill peptides prepared in Examples 1-3 and Comparative Examples 1-6

[0137]

[0138] As shown in Table 3, the Antarctic krill peptides prepared in Examples 1-3 have significantly better moisturizing and water-locking effects than those in Comparative Examples 1-6, and have broad application prospects in the cosmetics field.

Claims

1. A method for preparing Antarctic krill peptide using Lactobacillus plantarum, characterized in that: The following steps are involved: Step S1, defatting treatment: adding Antarctic krill powder to ethanol-water solution at a material-liquid ratio of 1:12-16, stirring for 6.2-8.4 hours, standing and filtering, drying the precipitate, crushing and sieving to obtain defatted krill powder; Step S2, sterilization treatment: adding defatted krill powder to deionized water, adjusting the pH to 8.2-8.6, sterilizing, and cooling to room temperature to obtain a sterilized krill culture medium; Step S3, fermentation: adding the activated composite seed bacterial solution and the fermentation synergist to the sterilized krill culture medium, stirring evenly, fermenting, centrifuging, collecting the supernatant, filtering, and homogenizing to obtain a fermentation suspension; Step S4, enzymatic hydrolysis: adding a composite enzyme and an enzymatic agent to the fermentation suspension, performing enzymatic hydrolysis to obtain an enzymatic fermentation extract, wherein the composite enzyme is prepared by mixing papain, alkaline protease, and composite protease in a mass ratio of 4:2-3:1, and the enzymatic agent is prepared by mixing calcium gluconate, N-butylpyridinium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous ethanol in a mass ratio of 20:80-100:5:7-8:1000-1200; Step S5, enzyme inactivation: inactivating the enzyme in the enzymatic fermentation extract, cooling, centrifuging, ultrafiltration, and freeze-drying to obtain Antarctic krill peptide; The activated composite seed bacterial liquid is prepared by the following steps: Step S11, using MRS broth as a basal medium, supplementing the basal medium with 2-3% carbon source, 0.01-0.03% magnesium sulfate, and 0.002-0.004% manganese sulfate, by mass percentage, and adjusting the pH value of the system to 5.2-5.6 to obtain an activated medium; Step S12, inoculating the composite bacteria into the activation culture medium obtained in step S11 at an inoculum amount of 6-8% by volume, setting the culture temperature to 25-30°C, shaking the culture on a shaker, adding an inorganic calcium source at the 4th and 6th hours of fermentation, the shaking rate is 100-200 rpm, and the culture time is 12-24 hours to obtain an activated composite seed bacterial solution, wherein the composite bacteria are obtained by mixing Lactobacillus plantarum F-B18-1 and Brevibacterium flavum at a viable cell count ratio of 1:0.3-0.5 in a sterile environment; The fermentation synergist is prepared by the following steps: Step A1, adding hydroxymethyl chitosan to an acetic acid aqueous solution, stirring until uniform, then adding sodium selenite, heating to 45-55° C., stirring and reacting for 2.2-2.8 hours, filtering, precipitating with alcohol, filtering, washing, drying, and grinding to obtain modified chitosan; Step A2: ultrasonically dispersing the modified chitosan in an acetic acid aqueous solution, adjusting the pH, filtering, adding a surfactant to the filtrate, stirring evenly, then adding fulvic acid, triacontanol, nicotinic acid, and brewer's yeast powder, and continuing to stir for 0.4-0.6 hours to obtain an emulsion; uniformly mixing the tripolyphosphate aqueous solution and the emulsion, and performing a cross-linking reaction for 0.35-0.55 hours. After the reaction is completed, centrifuging, washing, and then dispersing the centrifuged product in distilled water for ultrasonic treatment, and freeze-drying to obtain a fermentation enhancer.

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

2.

3. The method for preparing Antarctic krill peptide using Lactobacillus plantarum according to claim 1, characterized in that: In step S4, the mass ratio of the fermentation suspension, the compounded enzyme and the enzymatic agent is 10:0.24-0.26:0.06-0.

14.

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

5. The method for preparing Antarctic krill peptide using Lactobacillus plantarum according to claim 1, characterized in that: In step A2, the mass ratio of modified chitosan, acetic acid aqueous solution, surfactant, fulvic acid, triacontanol, nicotinic acid, brewer's 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.

6. Antarctic krill peptide prepared by the method for preparing Antarctic krill peptide by Lactobacillus plantarum according to any one of claims 1 to 5.

7. Use of the Antarctic krill peptide according to claim 6 in skin care products.

Images