Fermented Collagen Composition with Antioxidant and Moisturizing Functions and Preparation Method Thereof

Abstract

The present invention provides a preparation method of fermented collagen, comprising: S1) mixing collagen, a carbon source and water, and sterilizing to obtain a fermentation medium; S2) inoculating a Lactobacillus plantarum seed solution, a Lactobacillus rhamnosus seed solution, a Lactobacillus acidophilus seed solution, a Bifidobacterium lactis seed solution and a Lactobacillus bulgaricus seed solution into the fermentation medium for fermentation to obtain a fermentation broth; S3) sterilizing and filtering the fermentation broth to obtain fermented collagen. Compared with the prior art, the preparation method provided by the present invention ferments collagen by using a co-fermentation technology of multiple probiotics, reduces the molecular weight, effectively eliminates the fishy smell substances of collagen, so that the obtained fermented collagen has a high amino acid content, especially a high content of hydroxyl amino acids such as serine and threonine. It is not only beneficial to human absorption, improves the absorption efficiency and bioavailability, but also can improve the moisturizing effect of the product.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen, and particularly relates to a fermented collagen composition having antioxidant and moisturizing functions and a preparation method thereof. Background Art

[0002] Collagen is a high-molecular protein, accounting for more than 25% of the total protein in the human body, equivalent to 6% of the human body weight. It exists in human skin, bones and other parts, has the function of repairing injuries, maintains skin elasticity and luster, maintains a delicate and smooth appearance, and has skin repair function. Since collagen is converted into collagen peptides in the body and is directly and rapidly absorbed, with an absorption rate of more than 95%, it can also penetrate into tissues such as the skin, promoting the metabolism and regeneration of collagen, playing multiple roles such as moisturizing and antioxidant, so that collagen products are widely used in the fields of medicine, health products, cosmetics, etc.

[0003] At present, the raw materials of collagen products are basically the skins and bones of animals such as pigs and cows. However, due to reasons such as the mad cow disease panic, the use of collagen and its products is restricted. Now, fish skins and other waste from aquatic product processing have become ideal alternative raw materials. Moreover, China is rich in aquatic resources, and a large amount of fish skins, fish scales and other by-products are generated during aquatic product processing, which are rich in collagen and have become a research hotspot.

[0004] At present, the main preparation methods of polypeptides are: separation and extraction method, chemical synthesis method, gene recombination method and enzymatic hydrolysis of proteins method. The enzymatic hydrolysis method is one of the most studied methods at present, and it is the main method for producing bioactive peptides at present. This method has the advantages of high safety, low price and easy promotion.

[0005] For example, Chinese Patent with publication number CN101481723A discloses a method for preparing collagen peptides from carp skin, Chinese Patent with publication number CN101407834A discloses a method for preparing collagen peptides using cod skin, Chinese Patent with publication number CN10158227A discloses a process for producing collagen peptides from fish skin, "Study on the Optimal Conditions for Extracting Collagen Peptides from Tilapia Skin by Alkaline Protease Hydrolysis" (Food Research and Development, September 2008), etc.

[0006] However, the above studies still have the following problems: 1. The process is complex. When using fish skin to prepare collagen peptides, intermediate processes such as removing miscellaneous proteins and fats and further enzymatically hydrolyzing collagen to obtain collagen polypeptides are required. 2. The production cost is high. The proteases used in enzymatic hydrolysis are expensive, driving up the production cost. 3. The molecular weight of the obtained collagen peptides is large, mostly above 3000 Da, and the absorption effect is poor, unable to meet the requirements, reducing the product utilization rate.

[0007] The utilization of microorganisms by humans mainly has two stages: mixed culture and pure culture. Modern microbial fermentation is mostly pure fermentation, that is, a pure fermentation process using a single strain. The pure culture technology enables researchers to study a single strain without interference, thus enabling people to conduct deeper research on the physiological, biochemical, and genetic characteristics of microorganisms. However, many important biochemical processes cannot be completed or can only weakly react by a single strain of bacteria. Therefore, sometimes it is necessary to rely on the combined action of two or more microorganisms to complete, that is, mixed fermentation. Recently, scientific and technological workers have begun to use mixed cultures of microorganisms for fermentation to improve the deficiencies of single-strain fermentation. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a fermented collagen composition with antioxidant and moisturizing functions and a preparation method thereof. The fermented collagen prepared by this method, especially fermented fish skin collagen peptide, has a lower molecular weight and higher purity, which is more conducive to human absorption, thereby improving the absorption efficiency and bioavailability.

[0009] The present invention provides a method for preparing fermented collagen, comprising:

[0010] S1) Mix collagen, a carbon source, and water, and sterilize to obtain a fermentation medium;

[0011] S2) Inoculate Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus into the fermentation medium for fermentation to obtain a fermentation broth;

[0012] S3) After sterilizing and filtering the fermentation broth, obtain fermented collagen.

[0013] Preferably, the collagen is selected from collagen peptides; the molecular weight of the collagen peptide is 3000 - 10000 Da; the carbon source is selected from one or more of fructooligosaccharide, crystalline fructose, and granulated sugar.

[0014] Preferably, in step S1), the mass concentration of collagen in the fermentation medium is 5% - 20%; the mass concentration of the carbon source is 0.2% - 2%.

[0015] Preferably, the collagen is deep-sea cod collagen peptide; the pH value of the fermentation medium is 5.0 - 6.5;

[0016] In step S1), the sterilization temperature is 115°C - 121°C, and the sterilization time is 15 - 30 min;

[0017] In step S3), the sterilization temperature is 65°C - 75°C, and the sterilization time is 20 - 40 min.

[0018] Preferably, before inoculation, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus are activated respectively to obtain their respective seed liquids, and then inoculated into a fermentation medium;

[0019] The OD of the Lactobacillus plantarum seed liquid 600 is 2 - 6; the volume of the Lactobacillus plantarum seed liquid is 1% - 5% of the volume of the fermentation medium;

[0020] The OD of the Lactobacillus rhamnosus seed liquid 600 is 2 - 6; the volume of the Lactobacillus rhamnosus seed liquid is 1% - 5% of the volume of the fermentation medium;

[0021] The OD of the Lactobacillus acidophilus seed liquid 600 is 0.5 - 1; the volume of the Lactobacillus acidophilus seed liquid is 1% - 5% of the volume of the fermentation medium;

[0022] The OD of the Bifidobacterium lactis seed liquid 600 is 0.5 - 1; the volume of the Bifidobacterium lactis seed liquid is 1% - 5% of the volume of the fermentation medium;

[0023] The OD of the Lactobacillus bulgaricus seed liquid 600 is 2 - 6; the volume of the Lactobacillus bulgaricus seed liquid is 1% - 5% of the volume of the fermentation medium.

[0024] Preferably, after inoculating Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus into the fermentation medium in step S2), the pH value is adjusted to 5.5 - 6.5;

[0025] The temperature of the fermentation is 30°C - 40°C; the stirring speed during fermentation is 100 - 200 rpm; the tank pressure during fermentation is 0.02 - 0.1 MPa;

[0026] The fermentation time is 24 - 53 h; the pH value of the fermentation broth is 2.5 - 4.5.

[0027] Preferably, after sterilization in step S3), it is emulsified and homogenized, and then filtered; the emulsification and homogenization time is 25 - 50 min; the pore size of the filtration is 1 - 15 microns.

[0028] The present invention also provides a fermented collagen, which is prepared by fermenting collagen with a composite bacterium, and the composite bacterium includes Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus.

[0029] The present invention also provides a composite bacterium, including: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus.

[0030] The present invention also provides a collagen fermentation preparation, which includes the fermented collagen prepared by the above preparation method.

[0031] The present invention also provides an application of the fermented collagen prepared by the above preparation method in preparing a preparation with one or more functions of antioxidant, free radical scavenging, skin improvement, skin moisturizing, supplementing the serine content of the skin, and supplementing the threonine content of the skin.

[0032] The present invention provides a method for preparing fermented collagen, including: S1) mixing collagen, a carbon source and water, and sterilizing to obtain a fermentation medium; S2) inoculating a plant lactobacillus seed solution, a rhamnulose lactobacillus seed solution, an acidophilus lactobacillus seed solution, a bifidobacterium lactis seed solution and a bulgaricus lactobacillus seed solution into the fermentation medium for fermentation to obtain a fermentation broth; S3) sterilizing and filtering the fermentation broth to obtain fermented collagen. Compared with the prior art, the preparation method provided by the present invention ferments collagen by using a multi-probiotic co-fermentation technology, reduces the molecular weight, and effectively resolves the fishy smell substances of collagen, so that the obtained fermented collagen has a high amino acid content, especially a high content of hydroxyl amino acids such as serine and threonine. It is not only beneficial to human absorption, improves the absorption efficiency and bioavailability, but also can improve the moisturizing effect of the product.

[0033] Furthermore, the present invention provides a collagen fermentation preparation with excellent taste, rich in vitamins and extremely high nutritional value. Description of the Drawings

[0034] Figure 1 It is a column chart of the DPPH free radical scavenging rate;

[0035] Figure 2 It is a column chart of the reducing power determination experiment;

[0036] Figure 3 It is a comparison chart of the amino acid contents in the deep-sea cod collagen peptide concentrate and the fermented collagen in Example 2. Detailed Embodiments

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] The present invention provides a compound bacterium, including: plant lactobacillus, rhamnulose lactobacillus, acidophilus lactobacillus, bifidobacterium lactis and bulgaricus lactobacillus.

[0039] In the present invention, the above-mentioned Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus are preferably mixed in the form of seed solutions after being activated respectively, that is, mixed in the form of Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, Lactobacillus acidophilus seed solution, Bifidobacterium lactis seed solution and Lactobacillus bulgaricus seed solution; the volume ratio of the Lactobacillus plantarum seed solution, Lactobacillus rhamnosus seed solution, Lactobacillus acidophilus seed solution, Bifidobacterium lactis seed solution and Lactobacillus bulgaricus seed solution is preferably (0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5).

[0040] The OD of the Lactobacillus plantarum seed solution 600 is preferably 2 - 6; the OD of the Lactobacillus rhamnosus seed solution 600 is preferably 2 - 6; the OD of the Lactobacillus acidophilus seed solution 600 is preferably 0.5 - 1; the OD of the Bifidobacterium lactis seed solution 600 is preferably 0.5 - 1; the OD of the Lactobacillus bulgaricus seed solution 600 is preferably 2 - 6.

[0041] The present invention provides a preparation method of fermented collagen, comprising: S1) mixing collagen, a carbon source and water, and sterilizing to obtain a fermentation medium; S2) inoculating Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus into the fermentation medium for fermentation to obtain a fermentation broth; S3) sterilizing and filtering the fermentation broth to obtain fermented collagen.

[0042] Among them, the present invention does not have any special restrictions on the sources of all raw materials, and they can be commercially available.

[0043] Mix collagen, carbon source and water, and sterilize to obtain a fermentation medium; the collagen in the fermentation medium can not only provide a nitrogen source for the growth of the strain, but also be decomposed by the strain to obtain small-molecule fermented collagen; in the present invention, the collagen is preferably collagen peptide, more preferably fish skin collagen peptide, and still more preferably deep-sea cod collagen peptide; the molecular weight of the collagen peptide is preferably 3000-10000 Da; the mass concentration of collagen in the fermentation medium is preferably 5%-20%, more preferably 8%-20%, still more preferably 10%-20%, and most preferably 15%-20%; the carbon source can be any carbon source well-known to those skilled in the art without special limitation. For the convenience of the strain to utilize and grow, in the present invention, it is preferably to use one or more of fructooligosaccharide, crystalline fructose and granulated sugar as the carbon source; the mass concentration of the carbon source is preferably 0.2%-2%, more preferably 0.5%-1.5%, still more preferably 0.5%-1%; the sterilization temperature is preferably 115°C-121°C; the sterilization time is preferably 15-30 min; the pH value of the fermentation medium is preferably 5.0-6.5.

[0044] Inoculate Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus into the fermentation medium for fermentation to obtain a fermentation broth.

[0045] In the present invention, before inoculation, the above-mentioned Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus are preferably activated respectively to obtain their respective seed solutions, and inoculated in the form of seed solutions respectively.

[0046] Among them, the OD of the Lactobacillus plantarum seed solution 600 is preferably 2-6, more preferably 3-5, still more preferably 4-4.5; the volume of the Lactobacillus plantarum seed solution is preferably 1%-5% of the volume of the fermentation medium, more preferably 2%-4%, still more preferably 3%; the OD of the Lactobacillus rhamnosus seed solution 600 is preferably 2-6, more preferably 3-5, still more preferably 4-5, and most preferably 4.5-4.8; the volume of the Lactobacillus rhamnosus seed solution is preferably 1%-5% of the volume of the fermentation medium, more preferably 2%-4%, still more preferably 3%; the OD of the Lactobacillus acidophilus seed solution 600 is preferably 0.5-1, more preferably 0.6-0.9, still more preferably 0.7-0.8; the volume of the Lactobacillus acidophilus seed solution is preferably 1%-5% of the volume of the fermentation medium, more preferably 2%-4%, still more preferably 3%; the OD of the Bifidobacterium lactis seed solution 600Preferably 0.5 to 1, more preferably 0.6 to 0.9, still more preferably 0.6 to 0.8; the volume of the Bifidobacterium lactis seed liquid is preferably 1% to 5% of the volume of the fermentation medium, more preferably 2% to 4%, still more preferably 3%; the OD of the Lactobacillus delbrueckii subsp. bulgaricus seed liquid 600 is preferably 2 to 6, more preferably 3 to 6, still more preferably 4 to 5, most preferably 4.5 to 5; the volume of the Lactobacillus delbrueckii subsp. bulgaricus seed liquid is preferably 1% to 5% of the volume of the fermentation medium, more preferably 2% to 4%, still more preferably 3%.

[0047] The preparation methods of the above-mentioned seed liquids are all methods well-known to those skilled in the art, without special limitations, and preferably the seed liquids after secondary activation. In the present invention, it is preferably prepared according to the following method: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus delbrueckii subsp. bulgaricus are respectively inoculated into the first-stage seed medium and statically cultured to obtain the first-stage seed liquid; the first-stage seed medium is preferably MRS medium; the inoculation amounts of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus delbrueckii subsp. bulgaricus are preferably independently 1% to 3%; the temperature of the static culture is preferably 30°C to 40°C; the time of the static culture is preferably 12 to 16 h. The first-stage seed liquids of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus delbrueckii subsp. bulgaricus are respectively inoculated into the second-stage seed medium and stirred and cultured to obtain the second-stage seed liquid; the inoculation amounts of the first-stage seed liquids of Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus delbrueckii subsp. bulgaricus are preferably independently 1% to 5%, more preferably 2% to 4%, still more preferably 3%; the second-stage seed medium is preferably MRS medium; the temperature of the stirred culture is preferably 30°C to 40°C; the rotation speed of the stirred culture is preferably 100 to 200 rpm; the tank pressure of the stirred culture is preferably 0.01 to 0.1 MPa; the time of the stirred culture is preferably 10 to 15 h.

[0048] After inoculating the Lactobacillus plantarum seed liquid, Lactobacillus rhamnosus seed liquid, Lactobacillus acidophilus seed liquid, Bifidobacterium lactis seed liquid and Lactobacillus delbrueckii subsp. bulgaricus seed liquid into the fermentation medium, the pH value is preferably adjusted to 5.5 to 6.5, and then fermentation is carried out to obtain the fermentation broth; the temperature of the fermentation is preferably 30°C to 40°C; the stirring rotation speed during fermentation is preferably 100 to 200 rpm; the tank pressure during fermentation is preferably 0.02 to 0.1 MPa; the fermentation time is preferably 24 to 53 h; the pH value of the fermentation broth is preferably 2.5 to 4.5.

[0049] Sterilize the fermentation broth; the sterilization temperature is preferably 65°C to 75°C; the sterilization time is preferably 20 to 40 min.

[0050] After sterilization, it is preferably emulsified and homogenized and then filtered to obtain fermented collagen; the time for emulsification and homogenization is preferably 25 to 50 minutes; the filtration is preferably plate-and-frame filtration; the pore size of the filtration is preferably 1 to 15 microns.

[0051] The present invention uses multiple probiotics for fermentation, makes full use of the differences in the fermentation effects of different strains, obtains bioactive polypeptide substances, achieves the purpose of further decomposing collagen, reducing the molecular weight, and effectively resolving the fishy smell substances of collagen, so that the obtained fermented collagen has a high amino acid content, especially a high content of hydroxyl amino acids such as serine and threonine, which is not only beneficial to human absorption, improves the absorption efficiency and bioavailability, but also can improve the moisturizing effect of the product.

[0052] The present invention also provides a fermented collagen prepared by fermenting collagen with a complex bacterium; the complex bacterium includes Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus bulgaricus. The complex bacterium is the same as described above and will not be elaborated here; the collagen is preferably collagen peptide, more preferably fish skin collagen peptide, and still more preferably deep-sea cod collagen peptide; the molecular weight of the collagen peptide is preferably 3000 to 10000 Da.

[0053] The present invention also provides an application of the fermented collagen prepared by the above preparation method in the preparation of a preparation having one or more functions of antioxidant, free radical scavenging, skin improvement, skin moisturization, supplementing the serine content of the skin, and supplementing the threonine content of the skin.

[0054] The present invention also provides a collagen fermentation preparation, including the fermented collagen prepared by the above method.

[0055] To reconcile the taste of the collagen fermentation preparation, it preferably further includes concentrated fruit juice and / or sweetener.

[0056] In the embodiment provided by the present invention, the collagen fermentation preparation may be composed of the following components: 50 to 100 parts by weight of the above fermented collagen; 1 to 10 parts by weight of orange concentrated juice; 1 to 20 parts by weight of concentrated red date juice; 1 to 15 parts by weight of sweetener.

[0057] Among them, the orange concentrated juice is preferably blood orange concentrated juice; the sweetener is preferably erythritol.

[0058] The present invention also provides a preparation method of the above collagen fermentation preparation, including: mixing the fermented collagen, orange concentrated juice, concentrated red date juice, and sweetener, heating and dissolving them, and then performing heat sterilization treatment to obtain the collagen fermentation preparation.

[0059] Among them, the fermented collagen, orange concentrated juice, concentrated red date juice, and sweetener are all the same as described above and will not be elaborated here.

[0060] Mix the fermented collagen, concentrated orange juice, concentrated red date juice and sweetener, and heat and dissolve them; the temperature for heating and dissolving is preferably 50°C to 70°C, and the time for heating and dissolving is preferably 20 to 40 h.

[0061] Then raise the temperature for sterilization treatment to obtain a collagen fermentation preparation; the temperature for raising the temperature and sterilization treatment is preferably 90°C to 100°C; the time is preferably 15 to 30 min.

[0062] In order to further illustrate the present invention, the following is a detailed description of a fermented collagen composition with antioxidant and moisturizing functions and its preparation method provided by the present invention in conjunction with embodiments.

[0063] The reagents used in the following examples are all commercially available.

[0064] Example 1

[0065] 1.1 Preparation of fermentation medium: Add deep-sea cod collagen peptide with a molecular weight of 3000 Da and fructooligosaccharide to water to obtain a fermentation broth; the mass concentration of deep-sea cod collagen peptide in the fermentation broth is 15%, and the concentration of fructooligosaccharide is 0.5%; the fermentation broth is sterilized at 115 - 121°C for 15 - 30 min, and the parameters are adjusted (temperature 30 - 40°C; stirring speed 100 - 200 rpm; pH 5.0 - 6.5; tank pressure 0.02 - 0.10 MPa), and it is reserved for use.

[0066] 1.2 Preparation of seed liquid: Prepare a first-stage seed medium (MRS medium), 600 ml / bottle, 1 bottle, sterilize at 115 - 121°C for 15 - 30 min, and the pH is not controlled; inoculate Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, and Lactobacillus bulgaricus into the first-stage seed medium at an inoculation amount of 3% respectively; incubate statically at 30°C for 16 h, and reserve for use.

[0067] 1.3 Detection: Take samples and detect the seed liquid under a microscope to ensure that there is no contamination by miscellaneous bacteria, and detect the OD of the seed liquid 600 (OD of Lactobacillus plantarum 600 is 4.0; OD of Lactobacillus acidophilus 600 is 0.8; OD of Bifidobacterium lactis 600 is 0.6; OD of Lactobacillus rhamnosus 600 is 4.5; OD of Lactobacillus bulgaricus 600 is 5.0);

[0068] 1.4 Seed tank culture: Prepare the secondary seed culture medium (MRS medium), 20 L per tank, pH 6.0 ± 0.5, sterilize at 115 - 121 °C for 15 - 30 min; inoculate the cultured Lactobacillus plantarum and Lactobacillus acidophilus (inoculation amount is 3%) into seed tank 1, 600 ml per tank, inoculate the Lactobacillus rhamnosus seed liquid and Lactobacillus bulgaricus seed liquid (inoculation amount is 3%) into seed tank 2, 600 ml per tank; inoculate Bifidobacterium lactis (inoculation amount is 3%) into seed tank 3, 600 ml per tank. Maintain the fermentation parameters (temperature 30 - 40 °C; stirring speed 100 - 200 rpm; tank pressure 0.02 - 0.10 MPa), pH is not controlled, culture for 10 - 15 h;

[0069] 1.5 Detection: Sampling, microscopic detection of the seed liquid in the seed tank to ensure no contamination by miscellaneous bacteria, detect the OD of the seed liquid 600 (OD of Lactobacillus plantarum 600 is 4.5; OD of Lactobacillus acidophilus 600 is 0.7; OD of Bifidobacterium lactis 600 is 0.8; OD of Lactobacillus rhamnosus 600 is 4.8; OD of Lactobacillus bulgaricus 600 is 4.5);

[0070] 1.6 Set up the following groups and inoculate the strains of different groups into the fermentation medium for shake flask experiments respectively. The experimental conditions are temperature 37 °C; stirring speed 100 rpm; pH 6.5.

[0071] Group setting:

[0072] Experimental group: Five strains: Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus and Lactobacillus bulgaricus (inoculation amount is 3% for all).

[0073] Control group 1: Six strains: Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Streptococcus thermophilus (inoculation amount is 3% for all, and the activation method of Streptococcus thermophilus is the same as that of other strains, OD of the seed liquid 600 is 2.2).

[0074] Control group 2: Lactobacillus plantarum + Lactobacillus rhamnosus (inoculation amount is 3% for both).

[0075] Control group 3: Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus (inoculation amount is 3% for all).

[0076] Control group 4: Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus (inoculation amount is 3% for all).

[0077] Control group 5: Inoculation amount of Lactobacillus plantarum is 0.5%, inoculation amount of Lactobacillus rhamnosus is 3%, inoculation amount of Lactobacillus acidophilus is 3%, inoculation amount of Bifidobacterium lactis is 3%, inoculation amount of Lactobacillus bulgaricus is 3%.

[0078] Control group 6: Inoculation amount of Lactobacillus plantarum is 6%, inoculation amount of Lactobacillus rhamnosus is 3%, inoculation amount of Lactobacillus acidophilus is 3%, inoculation amount of Bifidobacterium lactis is 3%, inoculation amount of Lactobacillus bulgaricus is 3%.

[0079] Experiments were carried out using different strains and combinations, and by detecting indexes such as OD 600 and pH, the ability of each combination to ferment collagen was analyzed, and the results are shown in Table 1.

[0080] Table 1 Results of fermentation tests with different strains

[0081]

[0082] It can be seen from Table 1 that: 1) The experimental results at 24 h and 32 h of fermentation show that compared with the experimental group, OD 600 in control group 2, control group 3 and control group 4 are all lower than the latter, and the pH after fermentation is higher than that of the experimental group, indicating that the strain combination of the experimental group is more conducive to the fermentation of collagen.

[0083] 2) Streptococcus thermophilus was added to control group 1, the proportion of Lactobacillus plantarum in control group 5 was reduced to 0.5%, and the proportion of Lactobacillus plantarum in control group 6 was increased to 6%. Compared with the experimental group (a combination of five strains, the proportion of Lactobacillus plantarum is 3%), OD 600 are all lower than the latter, and the pH after fermentation is higher than that of the experimental group. It shows that compared with the experimental group, the bacterial growth in control group 1, control group 5 and control group 6 is better, and the ability to metabolize and produce acid is stronger, indicating that the experimental group (a combination of five strains) is more beneficial to the fermentation of collagen.

[0084] The results of the experimental group, control group 5 and control group 6 (all combinations of five strains) are better than those of control group 1 (a combination of six strains) and control group 4 (a combination of four strains). After the five strains are combined, the cell density is higher and the pH is lower than those of other groups, indicating better cell growth.

[0085] The DPPH free radical scavenging rate of the fermented collagen obtained by fermenting the above different strains was measured by DPPH analysis method to analyze the antioxidant ability of each combination after fermenting collagen.

[0086] Operation steps: Sample group A1: Take 2 ml of the sample solution and 2 ml of DPPH solution in a test tube, and shake well to mix evenly; Control group A2: Take 2 ml of the sample solution and 2 ml of absolute ethanol in a test tube, and shake well to mix evenly; Blank group A3: Take 2 ml of ultrapure water and 2 ml of DPPH solution in a test tube, and shake well to mix evenly;

[0087] React A1, A2, and A3 in the dark at room temperature for 30 min. Using absolute ethanol as the blank control, measure the absorbance at 517 nm, and repeat the experiment 3 times. Calculate using the following formula: The results obtained are shown in Table 2 and Figure 1 , Figure 1 is the column chart of DPPH radical scavenging rate. Among them, compared with the experimental group, **P < 0.01.

[0088] Table 2 Results of DPPH radical scavenging rate

[0089]

[0090] Statistical analysis was performed using SPSS 21.0 software. The data obtained from the experiment were expressed as mean Mean + SD, and the data of the experimental group were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

[0091] From Table 2 and Figure 1 it can be seen that the highest DPPH radical scavenging rate of the experimental group (five-strain combination) was 84.64%, which was higher than that of control groups 1 - 6, and there was a highly significant difference, P < 0.01. In this experiment, the experimental group (five-strain combination) had the strongest DPPH radical scavenging ability and antioxidant ability, and was most conducive to the fermentation of collagen.

[0092] The lowest DPPH radical scavenging rate of control group 2 (two-strain combination) was 68.47%. Control group 3 used (three-strain combination), control group 4 (four-strain combination), control groups 5 - 6 (five-strain combination), and the radical scavenging rates of control group 1 (six-strain combination) were all lower than those of the experimental group. The experiment on the radical scavenging rate confirmed that the five-strain combination of the present invention had a significant synergistic effect, and neither the increase nor the decrease of the strains could achieve the effect of the five-strain combination of the present invention.

[0093] Perform a reducing power determination test on the fermented collagen obtained by fermenting the above different strains. The operation steps are as follows:

[0094] Sample group Ax: Take 1 ml of the sample solution in a test tube, add 1 ml of potassium ferricyanide solution with a mass fraction of 1% and an equal volume of 0.2 mol / L phosphate buffer solution (pH 6.6). After mixing well, place the mixture in a constant temperature water bath at 50 °C and keep it static for 20 min. After taking it out, quickly cool it to room temperature. Then add 1 ml of trichloroacetic acid solution with a mass fraction of 10%. Shake it to mix completely. Place it in a high-speed centrifuge and centrifuge at 3000 r / min for about 10 min (or let it stand for 20 min). Take 1 ml of the supernatant after centrifugation in a test tube, add 3 ml of deionized water and 0.5 ml of ferric chloride solution with a mass fraction of 0.1%. Shake it to mix evenly. After standing and reacting at room temperature for 10 min, use ultrapure water as the blank and measure the absorbance at 700 nm. The magnitude of the absorbance is proportional to the reducing power of the sample.

[0095] Blank control group A0: Use an equal volume of deionized water to replace the sample solution as the blank control, and the method is the same as above.

[0096] The calculation formula is as follows: Reducing power OD = sample OD value - blank control OD value. The results are shown in Table 3 and Figure 2 , Figure 2 is the column chart for the reducing power determination experiment.

[0097] Table 3 Results of reducing power determination

[0098]

[0099]

[0100] Statistical methods were used for statistical analysis with SPSS 21.0 software. All the data obtained from the experiment were expressed as mean ± SD. The data of the experimental group were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant. + SD is used to represent, and the data of the experimental group were analyzed by one-way ANOVA (One-way ANOVA). P < 0.05 is statistically significant.

[0101] From Table 3 and Figure 2 it can be seen that: 1) The highest reducing power OD of the experimental group (five-strain combination) is 0.40997, which is higher than that of the control groups 1 - 6, and there is a significant difference, P < 0.05. The experimental results show that the fermentation experimental group (five-strain combination) has the strongest reducing ability, the strongest antioxidant ability, and is most conducive to the fermentation of collagen.

[0102] 2) The lowest fermentation reducing power of Control Group 2 (combination of two strains) was 0.2838. Control Group 3 used (combination of three strains), Control Group 4 (combination of four strains), Control Group 5 - 6 (combination of five strains), and the reducing power of Control Group 1 (combination of six strains) was lower than that of the experimental group. The experiment on reducing power confirmed that the combination of five strains of the present invention has a significant synergistic effect, and neither the increase nor the decrease of the strains can achieve the effect of the combination of five strains of the present invention.

[0103] In summary, the combination and inoculation ratio of five strains in the experimental group are significantly superior to other combinations or ratios in terms of the scavenging ability of DPPH and reducing power. The fermented collagen obtained using this combination has the strongest antioxidant ability.

[0104] Example 2

[0105] 2.1 Preparation of fermentation medium: Add deep - sea cod collagen peptide concentrate (concentration of the concentrate is 33%) and fructooligosaccharide solution (concentration is 55%) into water to obtain a fermentation broth; the mass concentration of deep - sea cod collagen peptide in the fermentation broth is 15%, and the concentration of fructooligosaccharide is 0.5%; the fermentation broth is sterilized at 121 °C for 20 min, and the parameters are adjusted (temperature 30 °C; stirring speed 100 rpm; pH 6.5; tank pressure 0.05 MPa), and it is reserved for use.

[0106] 2.2 Preparation of seed liquid: Prepare the first - stage seed MRS medium, 600 ml per bottle, 1 bottle, sterilize at 120 °C for 20 min, and the pH is not controlled; inoculate Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, and Lactobacillus bulgaricus into the first - stage seed medium at an inoculation amount of 3% respectively; culture statically at 30 °C for 16 h, and reserve for use.

[0107] 2.3 Detection: Sampling, microscopic detection of the seed liquid to ensure no contamination by miscellaneous bacteria, and detect the OD of the seed liquid 600 (OD of Lactobacillus plantarum 600 is 4.0; OD of Lactobacillus acidophilus 600 is 0.8; OD of Bifidobacterium lactis 600 is 0.6; OD of Lactobacillus rhamnosus 600 is 4.5; OD of Lactobacillus bulgaricus 600 is 5.0);

[0108] 2.4 Seed tank culture: Prepare the secondary seed MRS medium at 20 L per tank, with a pH of 6.0 ± 0.5, and sterilize it at 115 - 121 °C for 20 min; inoculate the well-cultured Lactobacillus plantarum and Lactobacillus acidophilus (inoculation amount 1 - 5%) into Seed Tank 1 at 600 ml per tank, inoculate the Lactobacillus rhamnosus seed liquid and Lactobacillus bulgaricus seed liquid (inoculation amount 1 - 5%) into Seed Tank 2 at 600 ml per tank; inoculate Bifidobacterium lactis (inoculation amount 1 - 5%) into Seed Tank 3 at 600 ml per tank, maintain the fermentation parameters (temperature 30 °C; stirring speed 100 rpm; tank pressure 0.05 MPa), do not control the pH, and culture for 12 h;

[0109] 2.5 Detection: Take samples and conduct microscopic detection of the seed liquid in the seed tank to ensure no contamination by miscellaneous bacteria, and detect the OD of the seed liquid 600 (OD of Lactobacillus plantarum 600 is 4.5; OD of Lactobacillus acidophilus 600 is 0.7; OD of Bifidobacterium lactis 600 is 0.8; OD of Lactobacillus rhamnosus 600 is 4.8; OD of Lactobacillus bulgaricus 600 is 4.5);

[0110] 2.6 Inoculate the fermenter: Inoculate the well-cultured seed liquid (Lactobacillus plantarum 3%, Lactobacillus rhamnosus 3%, Lactobacillus acidophilus 3%, Bifidobacterium lactis 3%, Lactobacillus bulgaricus 3% (V / V)) into the fermenter, with the volume of the fermentation broth being 200 L per tank. After inoculation, adjust the pH to 6.5, and do not control the pH during the subsequent fermentation. Maintain the fermentation parameters (temperature 37 °C; stirring speed 100 rpm; tank pressure 0.05 MPa)

[0111] 2.7 Fermentation end: Regularly take samples to detect the fermentation broth. When the fermentation time is 45 h ± 8 and the pH = 3.0 ± 0.5, the fermentation ends;

[0112] 2.8 Sterilization: Raise the temperature to 72 °C and treat for 30 min;

[0113] 2.9 Emulsification and homogenization (30 min)

[0114] 2.10 Plate and frame filtration (10 - micron pore size) to obtain fermented collagen.

[0115] Determine the amino acid content in deep-sea codfish collagen peptide and the fermented collagen obtained in Example 2 (in accordance with GB5009.124 - 2016), and obtain the comparison chart as Figure 3 shown.

[0116] From Figure 3It can be seen that the content of the *-labeled amino acids increases after fermentation. Among them, the content of Ser (serine) increases by 71.43% after fermentation, and the content of Thr (threonine) increases by 15.38%. Serine is one of the important natural moisturizing factors. It can increase the vitality and moisture retention of epidermal cells, delay skin aging, and is the main role in maintaining the moisture of the skin cutin layer. It also has antibacterial, buffering and surfactant protection effects. It can improve skin elasticity, strengthen nutrition, and maintain the vitality of surface cells. It is a key additive in many high-grade cosmetics. Threonine (Thr) is an important nutritional fortifier, which has the effect of relieving human fatigue and promoting growth and development. In medicine, due to the presence of a hydroxyl group in the structure of threonine, it has a water-holding effect on human skin and plays an important role in protecting cell membranes when combined with oligosaccharide chains.

[0117] Conclusion: The content of hydroxyl amino acids such as serine and threonine increases significantly after fermentation. It can be inferred that the effects of fermented collagen on skin moisturizing, protection and whitening are improved compared with those before fermentation.

[0118] Example 3

[0119] Mix 1-6% frozen blood orange concentrate, 2-10% concentrated red date juice, 1-8% erythritol (powder), and 80-90% of the fermented collagen obtained in Example 2 at 60°C and disperse them fully for 30 h to dissolve; heat up to 90°C and keep warm for 15-30 min to obtain a collagen fermentation preparation, and measure its pH (pH 4.0 ± 0.5) and soluble solids (≥15%).

Claims

1. A preparation method of fermented collagen, characterized in that, Comprising: S1) Mix collagen, carbon source and water, and sterilize to obtain a fermentation medium; S2) Inoculate Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus into the fermentation medium for fermentation to obtain a fermentation broth; S3) Sterilize and filter the fermentation broth to obtain fermented collagen; The collagen is selected from collagen peptides; the carbon source is selected from one or more of fructooligosaccharide, crystalline fructose and white granulated sugar; Before inoculation, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus are respectively activated to prepare their respective seed solutions, and then inoculated into the fermentation medium; OD of Lactobacillus plantarum seed liquid 600 is 2 to 6; the volume of the Lactobacillus plantarum seed liquid is 1% to 5% of the volume of the fermentation medium; OD of Lactobacillus rhamnosus seed solution 600 is 2-6; the volume of the Lactobacillus rhamnosus seed solution is 1%-5% of the volume of the fermentation medium; OD of Lactobacillus acidophilus seed solution 600 is 0.5 to 1; the volume of the Lactobacillus acidophilus seed solution is 1% to 5% of the volume of the fermentation medium; OD of the Bifidobacterium lactis seed solution 600 is 0.5 to 1; the volume of the Bifidobacterium lactis seed solution is 1% to 5% of the volume of the fermentation medium; OD of Lactobacillus bulgaricus seed solution 600 is 2 to 6; the volume of the Lactobacillus bulgaricus seed solution is 1% to 5% of the volume of the fermentation medium.

2. The preparation method according to claim 1, wherein The molecular weight of the collagen peptide is 3000 - 10000 Da; In step S1), the mass concentration of collagen in the fermentation medium is 5% - 20%; the mass concentration of the carbon source is 0.2% - 2%.

3. The preparation method according to claim 1, characterized in that, The collagen is deep - sea cod collagen peptide; the pH value of the fermentation medium is 5.0 - 6.5; In step S1), the sterilization temperature is 115°C - 121°C, and the sterilization time is 15 - 30 min; In step S3), the sterilization temperature is 65°C - 75°C, and the sterilization time is 20 - 40 min.

4. The preparation method according to claim 1, wherein In step S2), after inoculating Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus bulgaricus into the fermentation medium, adjust the pH value to 5.5 - 6.5; The fermentation temperature is 30°C - 40°C; the stirring speed during fermentation is 100 - 200 rpm; the tank pressure during fermentation is 0.02 - 0.1 MPa; The fermentation time is 24 - 53 h; the pH value of the fermentation broth is 2.5 - 4.

5.

5. The preparation method according to claim 1, characterized in that, In step S3), after sterilization, perform emulsification and homogenization, and then filter; the emulsification and homogenization time is 25 - 50 min; the pore size of the filter is 1 - 15 microns.

6. A collagen fermentation preparation, characterized in that, Comprising the fermented collagen prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the fermented collagen prepared by the preparation method according to any one of claims 1 - 5 in the preparation of a preparation with antioxidant function.

8. Use of the fermented collagen prepared by the preparation method according to any one of claims 1 - 5 in the preparation of a preparation with skin moisturizing function.

9. Use of the fermented collagen prepared by the preparation method according to any one of claims 1 - 5 in the preparation of a preparation with skin - improving function.

10. Use of the fermented collagen prepared by the preparation method according to any one of claims 1 - 5 in the preparation of a preparation with the function of supplementing the serine content of the skin.

11. Use of the fermented collagen prepared by the preparation method according to any one of claims 1 - 5 in the preparation of a preparation with the function of supplementing the threonine content of the skin.

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