Preparation method of filtrate of fermentation product of Bifidobacterium longum and its lysate

Abstract

The present invention discloses a preparation method of a filtrate of fermentation products of Bifidobacterium longum and its lysate, which relates to the technical field of microorganisms. A strain of Bifidobacterium longum CGMCC No. 31133 with fast growth rate, high cell density, rich and high-content metabolites is obtained through strain mutagenesis and breeding. The strain is used for fermenting to prepare a filtrate of fermentation products and a lysate. The filtrate is rich in acetic acid, lactic acid, amino acids, proteins and Bifidobacterium longum polysaccharides, and has remarkable skin brightening and moisturizing effects. The alkali lysis method is used for cell wall breaking, which not only makes the release of intracellular active substances more complete, but also effectively utilizes the active components of cell walls and cell membranes. The prepared lysate has richer active components and is easy to absorb, further enhancing the antioxidant, anti-aging, skin brightening and repair performance of the lysate. The filtrate of fermentation products and the lysate prepared by the present invention not only have rich active components and excellent performance, but also have a simple process and a short production cycle, and have significant cost advantages in production.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a preparation method of a filtrate of Bifidobacterium longum fermentation products and its lysate. Background Art

[0002] The skin is the first barrier of the human body. Healthy skin can resist external pollution, irritation, damage and sunlight, and also has moisturizing and regulating functions. However, daily environmental problems such as wind, sun, air pollution, etc. can damage the skin, leading to skin problems such as oxidation, aging, acne, dullness, pigmentation, wrinkles, etc. Bifidobacterium longum is an important intestinal probiotic, which is abundantly present in the intestines of breastfed infants. It has many important physiological functions for human health, such as biological barrier, nutritional function, anti-tumor function, immune enhancement function, improvement of gastrointestinal function, anti-aging, etc., and has great market prospects. The filtrate of Bifidobacterium longum fermentation products mainly refers to the filtrate after centrifugation or membrane filtration to remove bacteria from the fermentation of Bifidobacterium longum; while the lysate of Bifidobacterium longum refers to the intracellular products obtained by ultrasonic, high-pressure homogenization, alkaline lysis or enzymatic lysis of the fermented Bifidobacterium longum cells.

[0003] Due to the limitations of the production process, there are still many deficiencies in the existing production technologies of Bifidobacterium longum fermentation product filtrate and lysate, which are mainly reflected in:

[0004] 1. The utilization of Bifidobacterium longum fermentation products is single, and the high-value utilization of Bifidobacterium longum fermentation products has not been realized. At present, the utilization of Bifidobacterium longum fermentation products either only uses its lysate (patent applications CN201210012250.8, CN202010315316.5, CN 202010763305.3, CN 202111266902.6), or only uses the filtrate (patent applications CN202110985291.4, CN202111249067.5), or mixes the lysate and the filtrate only as one product (patent applications CN 202110166914.5, CN 202110941415.9); this is mainly because the effective active ingredients in the filtrate are relatively single and the performance is not obvious, resulting in it being unable to be presented as a product alone. Therefore, there are few reports on the simultaneous preparation of filtrate and lysate products, and the high-value utilization of Bifidobacterium longum fermentation products has not been well realized.

[0005] 2. There are few reports on the component analysis and performance research of the filtrate of Bifidobacterium longum fermentation products. Although there are reports on the production of the filtrate of Bifidobacterium longum fermentation products, the active components of the filtrate have not been analyzed, and its performance has not been studied in detail. Although patent application CN202310392623.7 has studied the application of the filtrate in inhibiting melanin, the research on its skin brightening and moisturizing properties, which are the most widely used in the cosmetics field, has not been carried out, lacking effective efficacy data.

[0006] 3. The nutrient components of the lysate are single, and the efficacy of the Bifidobacterium longum lysate cannot be maximally exerted. At present, the preparation methods of Bifidobacterium longum lysate mainly include ultrasonic cell wall breaking (CN201210012250.8), enzymatic hydrolysis and thermal lysis methods (CN202010315316.5), high-pressure homogenization cell wall breaking treatment (CN 202010763305.3, CN 202111266902.6, CN202110941415.9), mechanical crushing method and protease digestion method (CN 202110166914.5). However, these cell wall breaking methods only utilize the active components of intracellular products, and the active components of cell walls and cell membranes, which account for about 40-60% of the cell weight, are not fully utilized, resulting in a relatively single composition of the lysate and unable to exert the maximum performance of the lysate product.

[0007] 4. The production cost is high. Traditional lysate preparation methods such as ultrasonic crushing, high-pressure homogenization, mechanical crushing, and enzymatic lysis have the problems of high equipment investment, the need for ice bath during cell crushing, and the need for centrifugation to remove cell debris after crushing. The process operation is complex, resulting in high production costs and restricting the application and promotion of Bifidobacterium longum fermentation products. Summary of the Invention

[0008] In view of the problems existing in the current Bifidobacterium longum fermentation products, the present invention proposes the following solutions: (1) Prepare the filtrate and its lysate to highly utilize the Bifidobacterium longum fermentation products; (2) Analyze the active components of the fermentation product filtrate and study its skin brightening and moisturizing properties; (3) Use the alkali lysis method to extract the lysate, which not only fully utilizes intracellular products but also makes full use of the active components of cell walls and cell membranes, increases the content of active components in the lysate, and enhances the biological activity of the lysate; (4) Use the alkali lysis method to extract the lysate, which avoids equipment investment and does not require centrifugation to remove cell debris, reduces equipment investment and shortens the lysate preparation process, and effectively reduces the production cost. The purpose of the present invention is to solve the existing problems of Bifidobacterium longum fermentation products and provide a preparation method that can greatly improve the efficacy and economic benefits of the filtrate and lysate of Bifidobacterium longum fermentation products.

[0009] The overall technical concept of the present invention is:

[0010] Preparation method of filtrate and lysate of fermentation product of Bifidobacterium longum, the method comprising the following technological steps:

[0011] (1) Inoculating Bifidobacterium longum ( Bifidobacterium longum ) strain CGMCC No.31133 into slant medium in a test tube to prepare slant strains;

[0012] (2) Subculturing the slant strains for expansion as seed liquid;

[0013] (3) Inoculating the seed liquid into a fermenter for fermentation;

[0014] (4) Preparation of filtrate of fermentation product;

[0015] (5) Preparation of lysate of Bifidobacterium longum.

[0016] The specific technological steps and technological conditions of the present invention are:

[0017] In step (1), the slant medium consists of the following components in parts by weight:

[0018] Glucose 10 - 30 parts, tryptone 1 - 5 parts, beef peptone 1 - 5 parts, yeast extract powder 1 - 5 parts, sodium acetate 1 - 5 parts, diammonium hydrogen citrate 1 - 2 parts, dipotassium hydrogen phosphate 1 - 2 parts, agar 15 parts, and adding water to make up to 1000 parts;

[0019] The technological conditions for preparing slant strains are: culture temperature 30 - 40 °C, culture time 4 - 24 hours.

[0020] In step (2), the seed liquid and in step (3), the fermentation broth consist of the following components in parts by weight:

[0021] Glucose 10 - 30 parts, tryptone 1 - 5 parts, beef peptone 1 - 5 parts, yeast extract powder 1 - 5 parts, sodium acetate 1 - 5 parts, diammonium hydrogen citrate 1 - 2 parts, dipotassium hydrogen phosphate 1 - 2 parts, and water 1000 parts;

[0022] The culture conditions of the seed liquid are: culture temperature 30 - 40 °C, static and airtight culture, culture time 4 - 18 hours; then inoculating into a fermenter, and the fermentation technological conditions of the fermenter are: inoculation amount 10%, fermentation temperature 30 - 40 °C, static and airtight, fermentation time 2 - 8 hours, the acetic acid content in the filtrate of Bifidobacterium longum fermentation product is 5 - 20 g / L, the lactic acid content is 10 - 60 g / L, the amino acid content is 0.5 - 10 g / L, the protein content is 0.1 - 5 g / L, the polysaccharide content is 0.1 - 20 g / L; the wet weight of Bifidobacterium longum cells is 20 - 150 g / L.

[0023] The preparation of the fermentation product filtrate in step (4) includes the following three steps:

[0024] a. Centrifuge the fermented broth after fermentation to remove the thalli using a disc centrifuge;

[0025] b. Remove impurities such as pigments from the supernatant after step a using activated carbon;

[0026] c. Desalt the decolorized solution after step b to obtain a colorless and transparent filtrate product of the fermentation product of Bifidobacterium longum.

[0027] The preparation of the Bifidobacterium longum cell lysate in step (5) includes the following three steps:

[0028] a. Obtain the thallus cells by centrifuging the fermented broth;

[0029] b. Add the thallus cells after step a to an alkaline solution for alkaline lysis to obtain a cell lysate;

[0030] c. Desalt the lysate after step b to finally obtain a Bifidobacterium longum cell lysate product with an amino acid content of 0.5 - 20 g / L, a protein content of 0.5 - 20 g / L, and a polysaccharide content of 0.1 - 20 g / L.

[0031] The Bifidobacterium longum Bifidobacterium longum strain CGMCC No.31133 used in the present invention was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, No. 3, Beichen West Road, Chaoyang District, Beijing on July 2, 2024, with the deposit number being Bifidobacterium longum CGMCC No.31133.

[0032] The screening of the Bifidobacterium longum strain CGMCC No.31133 consists of the following technological steps:

[0033] (1) Sampling and enrichment culture;

[0034] (2) Subject the enriched strains to compound mutagenesis;

[0035] (3) Subject the mutagenized strains to fermentation screening;

[0036] (4) Conduct a subculture stability experiment on the selected strains.

[0037] The specific technological steps and process conditions are as follows:

[0038] Step (1) Sampling and enrichment culture:

[0039] Take a small amount of sample from infant feces, dissolve it in sterilized physiological saline, then spread it on a plate for enrichment culture. Select colonies that are white, round and convex, with neat edges and smooth surfaces, and conduct microscopic examination. The cells are generally 0.9 - 1.2 μm in length and 0.4 - 0.6 μm in width, showing irregular shapes such as Y-shaped, V-shaped or curved, without flagella and spores. Ferment the strain, sequence the genes of the cells, and compare them with the gene sequence of Bifidobacterium longum to determine that the starting strain is Bifidobacterium longum.

[0040] The enrichment plate medium consists of the following components in parts by weight:

[0041] 10 - 30 parts of glucose, 1 - 5 parts of tryptone, 1 - 5 parts of beef peptone, 1 - 5 parts of yeast extract powder, 1 - 5 parts of sodium acetate, 1 - 2 parts of diammonium hydrogen citrate, 1 - 2 parts of dipotassium hydrogen phosphate, 15 parts of agar, and add water to make up to 1000 parts;

[0042] The process conditions for preparing the plate are a temperature of 30 - 40°C and a culture time of 4 - 24 hours;

[0043] Step (2) Composite mutagenesis of the strain:

[0044] The composite mutagenesis sequentially includes the preparation of the seed bacterial suspension, the addition amount of the mutagen, and the UV irradiation time.

[0045] Inoculate the strain obtained in step (1) into a test tube containing 5 mL of physiological saline, add a mutagen of diethyl sulfate with a volume percentage of 0.5% - 2.0% to the test tube, shake it on a vortex oscillator for 1 - 2 min, take 0.5 mL of the bacterial suspension and spread it on the solid plate in step (1), irradiate the plate with a UV lamp for 20 - 60 s, and then incubate the plate in the dark. The culture conditions are: a culture temperature of 30 - 40°C and a culture time of 4 - 24 hours;

[0046] Step (3) Screening of the strain:

[0047] The screening fermentation broth consists of the following components in parts by weight:

[0048] 10 - 30 parts of glucose, 1 - 5 parts of tryptone, 1 - 5 parts of beef peptone, 1 - 5 parts of yeast extract powder, 1 - 5 parts of sodium acetate, 1 - 2 parts of diammonium hydrogen citrate, 1 - 2 parts of dipotassium hydrogen phosphate and 1000 parts of water; The fermentation process conditions are: a culture temperature of 30 - 40°C, static sealed fermentation, and a fermentation time of 4 - 24 hours; Screen strains with fast growth, high cell density, high lactic acid and acetic acid content, and the ability to metabolize biological polysaccharides (named Bifidobacterium longum polysaccharide);

[0049] Step (4) Subculture stability of the strain:

[0050] The selected strains are continuously cultured for more than 8 generations, and each generation is fermented. Strains with stable growth rate, cell density, and metabolite content in the 8th generation are considered as industrial production strains.

[0051] Finally, Bifidobacterium longum strains with fast growth rate, high cell density, and metabolites such as acetic acid, lactic acid, amino acids, and proteins, and which can produce high levels of Bifidobacterium longum polysaccharide are obtained Bifidobacterium longum ), namely, Bifidobacterium longum (

[0052] The technological progress achieved by the present invention lies in:

[0053] 1. Making full use of the fermentation product filtrate and lysate of Bifidobacterium longum to achieve high-value utilization of fermentation products:

[0054] In the present invention, Bifidobacterium longum is used for fermentation, and then the fermentation product filtrate is obtained by centrifugation; the collected bacterial cells are lysed by alkali to obtain the lysate; and according to the application, the filtrate and the lysate can be mixed in a certain proportion to obtain a lysate-filtrate composite product, making full use of the fermentation products of Bifidobacterium longum and achieving high-value utilization of fermentation products.

[0055] 2. The fermentation product filtrate is rich in active ingredients, has excellent moisturizing performance and obvious skin brightening effect:

[0056] (1) Through the analysis of the main active ingredients of the fermentation product filtrate obtained in the present invention, it is found that in addition to being rich in active ingredients such as acetic acid (5 - 20 g / L), lactic acid (10 - 60 g / L), amino acids (0.5 - 10 g / L), and proteins (0.1 - 5 g / L), the filtrate also contains novel Bifidobacterium longum polysaccharide (0.1 - 20 g / L). There are no relevant literature reports on the fermentation product filtrate of Bifidobacterium longum with such rich and high-content active ingredients.

[0057] (2) Excellent skin brightening and moisturizing performance:

[0058] The Bifidobacterium longum strains selected in the present invention can metabolize active ingredients such as acetic acid, lactic acid, amino acids, and proteins, which can significantly inhibit the activity of tyrosinase and have excellent skin brightening effects; in addition, the filtrate also contains novel Bifidobacterium longum polysaccharide, effectively enhancing the moisturizing performance of the filtrate product; further expanding the application field of the fermentation product filtrate and improving the product performance and its value.

[0059] (3) The skin feel of the filtrate containing Bifidobacterium longum polysaccharide is good: In addition to containing conventional active ingredients such as acetic acid, lactic acid, amino acids, and proteins, the fermentation product filtrate of the present invention is also rich in Bifidobacterium longum polysaccharide, having a refreshing and skin-moisturizing feel, providing a better product experience for consumers, and promoting the application of the fermentation product filtrate in the daily chemical field.

[0060] 3. The lysate has rich and high-content active ingredients and more excellent performance:

[0061] (1) The present invention uses an alkali lysis method for cell wall breaking. Compared with traditional ultrasonic wall breaking, high-pressure homogenization wall breaking, mechanical crushing method, and lysozyme lysis method, it has the following advantages: ① The alkali lysis method makes the cell wall breaking more complete, increases the dissolution ratio of cell contents, and improves the preparation efficiency of the lysate of Bifidobacterium longum; ② Alkali lysis can fully hydrolyze the cell wall and cell membrane accounting for 40-60% of the cell weight into choline, glucan, chitosan, acidic polysaccharide, peptidoglycan, etc. for utilization, greatly enriching the active ingredients of the lysate and laying a foundation for the preparation of high-performance Bifidobacterium longum lysate. There is no relevant literature report on the preparation of Bifidobacterium longum lysate using the alkali lysis method; ③ Alkali lysis can hydrolyze macromolecular proteins into small peptides and amino acids, which is more conducive to the absorption and utilization by human skin and further improves the skin care performance of the lysate.

[0062] (2) Excellent antioxidant, anti-aging, brightening, and repair performance: Since the lysate obtained by alkali lysis has richer and higher-content active ingredients, its antioxidant, anti-aging, brightening, and repair effects have been greatly improved, solving the problems of single composition and poor skin absorption of traditional Bifidobacterium longum lysate, and effectively improving the quality of the lysate product.

[0063] 4. Low production cost of the lysate: The present invention using alkali lysis can reduce equipment investment; after lysis, there is no need to centrifuge to remove cell wall fragments, thus shortening the process flow; the fermentation cycle of the present invention is 4-6 hours, significantly shortening the production cycle of the filtrate of Bifidobacterium longum fermentation product and its lysate, effectively saving the product production cost, and laying a foundation for large-scale and low-cost production of Bifidobacterium longum fermentation product and its lysate. Description of the Drawings

[0064] Figure 1 It is the infrared spectrogram of Bifidobacterium longum fermentation product filtrate and Bifidobacterium longum polysaccharide. Detailed Embodiments

[0065] Example 1. Preparation of the filtrate of Bifidobacterium longum CGMCC No. 31133 fermentation product and its lysate

[0066] (1) Slant culture of the strain

[0067] Inoculate the Bifidobacterium longum CGMCC No. 31133 strain into the slant medium in a test tube to make a slant strain;

[0068] Among them, the slant medium of the strain is composed of the following components by weight:

[0069] 20 parts of glucose, 2.5 parts of tryptone, 2.5 parts of beef peptone, 2.5 parts of yeast extract powder, 2 parts of sodium acetate, 1 part of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate, 15 parts of agar, and add water to make up to 1000 parts;

[0070] The technological conditions for preparing slant strains are: culture temperature 37 °C, culture time 24 hours.

[0071] (2)Seed liquid fermentation

[0072] Expand the culture of the slant strain as the seed liquid;

[0073] Among them, the seed liquid medium consists of the following components by weight:

[0074] 20 parts of glucose, 2.5 parts of tryptone, 2.5 parts of beef peptone, 2.5 parts of yeast extract powder, 2 parts of sodium acetate, 1 part of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate and 1000 parts of water;

[0075] The technological conditions for fermentation are: culture temperature 37 °C, static and airtight fermentation, fermentation time 16 hours.

[0076] (3)Fermentation in fermenter

[0077] Inoculate the seed liquid into the fermenter for fermentation;

[0078] Among them, the fermentation broth consists of the following components by weight:

[0079] 20 parts of glucose, 2.5 parts of tryptone, 2.5 parts of beef peptone, 2.5 parts of yeast extract powder, 2 parts of sodium acetate, 1 part of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate and 1000 parts of water;

[0080] The technological conditions for fermentation in the fermenter are: inoculum size 10%, fermentation temperature 37 °C, static and airtight, fermentation time 4 hours, and after fermentation, prepare the filtrate of the fermentation product and the cell lysate.

[0081] (4)Preparation of the filtrate of the fermentation product, including the following three steps:

[0082] a. Centrifuge the fermented broth after fermentation to remove the thallus with a disc centrifuge;

[0083] b. Remove impurities such as pigments from the supernatant after step a with activated carbon;

[0084] c. Desalt the decolorized solution after step b to obtain a colorless and transparent filtrate product of Bifidobacterium longum fermentation product.

[0085] (5)Preparation of the cell lysate of Bifidobacterium longum includes the following three steps:

[0086] a. Wash the bacterial cells obtained by centrifuging the fermentation broth twice with pure water to remove the components of the fermentation broth on the cell surface;

[0087] b. Subject the bacterial cells after step a to alkaline lysis to obtain a cell lysate;

[0088] c. Desalt the lysate after step b to finally obtain a product of Bifidobacterium longum lysate.

[0089] Example 2. Preparation of the filtrate of the fermentation product of Bifidobacterium longum CGMCC No. 31133 and its lysate

[0090] (1) Slant culture of the strain

[0091] Inoculate the Bifidobacterium longum CGMCC No. 31133 strain into the slant medium in a test tube to prepare a slant strain;

[0092] Among them, the slant medium of the strain consists of the following components in parts by weight:

[0093] Glucose 10 parts, tryptone 1 part, beef peptone 1 part, yeast extract powder 1 part, sodium acetate 2 parts, diammonium hydrogen citrate 2 parts, dipotassium hydrogen phosphate 1 part, agar 15 parts, and make up to 1000 parts with water;

[0094] The technological conditions for preparing the slant strain are: culture temperature 37 °C, culture time 24 hours.

[0095] (2) Fermentation of the seed liquid

[0096] Expand the culture of the slant strain as the seed liquid;

[0097] Among them, the seed liquid medium consists of the following components in parts by weight:

[0098] Glucose 10 parts, tryptone 5 parts, beef peptone 5 parts, yeast extract powder 1 part, sodium acetate 1 part, diammonium hydrogen citrate 1 part, dipotassium hydrogen phosphate 2 parts, and water 1000 parts;

[0099] The technological conditions for fermentation are: culture temperature 37 °C, static sealed fermentation, fermentation time 14 hours.

[0100] (3) Fermentation in a fermenter

[0101] Inoculate the seed liquid into the fermenter for fermentation;

[0102] Among them, the fermentation broth consists of the following components in parts by weight:

[0103] Glucose 10 parts, tryptone 5 parts, beef peptone 5 parts, yeast extract powder 1 part, sodium acetate 1 part, diammonium hydrogen citrate 1 part, dipotassium hydrogen phosphate 2 parts, and water 1000 parts;

[0104] The fermentation process conditions in the fermenter are as follows: inoculum size 10%, fermentation temperature 37°C, static and airtight, fermentation time 6 hours, and after fermentation, the preparation of fermentation product filtrate and lysate is carried out.

[0105] (4) The preparation of the fermentation product filtrate includes the following three steps:

[0106] a. Centrifuge the fermented broth after fermentation to remove the bacteria by disc centrifuge;

[0107] b. Remove impurities such as pigments from the supernatant after step a with activated carbon;

[0108] c. Desalt the decolorized solution after step b to obtain a colorless and transparent filtrate product of Bifidobacterium longum fermentation product.

[0109] (5) The preparation of the lysate of Bifidobacterium longum includes the following three steps:

[0110] a. Wash the bacterial cells obtained by centrifuging the fermented broth twice with pure water to remove the components of the fermented broth on the cell surface;

[0111] b. Alkaline lyse the bacterial cells after step a to obtain cell lysate;

[0112] c. Desalt the lysate after step b to finally obtain a Bifidobacterium longum lysate product.

[0113] Example 3. Preparation of the filtrate of Bifidobacterium longum CGMCC No. 31133 fermentation product and its lysate

[0114] (1) Inclined plane culture of the strain

[0115] Inoculate the Bifidobacterium longum CGMCC No. 31133 strain into the inclined plane medium in a test tube to make an inclined plane strain;

[0116] Among them, the inclined plane medium of the strain is composed of the following components by weight:

[0117] 30 parts of glucose, 1 part of tryptone, 1 part of beef peptone, 5 parts of yeast extract powder, 5 parts of sodium acetate, 2 parts of diammonium citrate, 1 part of dipotassium hydrogen phosphate, 15 parts of agar, and make up to 1000 parts with water;

[0118] The process conditions for preparing the inclined plane strain are: culture temperature 37°C, culture time 24 hours.

[0119] (2) Seed liquid fermentation

[0120] Expand the culture of the inclined plane strain as the seed liquid;

[0121] Among them, the seed liquid medium is composed of the following components by weight:

[0122] 30 parts of glucose, 1 part of tryptone, 1 part of beef peptone, 5 parts of yeast extract powder, 5 parts of sodium acetate, 2 parts of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate and 1000 parts of water;

[0123] The fermentation process conditions are: the culture temperature is 37 °C, static and sealed fermentation, and the fermentation time is 12 hours.

[0124] (3)Fermentation in a fermenter

[0125] Inoculate the seed liquid into the fermenter for fermentation;

[0126] Among them, the fermentation broth consists of the following components in parts by weight:

[0127] 30 parts of glucose, 1 part of tryptone, 1 part of beef peptone, 5 parts of yeast extract powder, 5 parts of sodium acetate, 2 parts of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate and 1000 parts of water;

[0128] The fermentation process conditions in the fermenter are: the inoculation amount is 10%, the fermentation temperature is 37 °C, static and sealed, the fermentation time is 3 hours, and after fermentation, the fermentation product filtrate and the lysate are prepared.

[0129] (4)Preparation of the fermentation product filtrate, including the following three steps:

[0130] a. Centrifuge the fermented broth after fermentation to remove the bacteria;

[0131] b. Activated carbon is used to remove impurities such as pigments from the supernatant after step a;

[0132] c. Desalt the decolorized liquid after step b to obtain a colorless and transparent filtrate product of Bifidobacterium longum fermentation product.

[0133] (5)Preparation of the lysate of Bifidobacterium longum includes the following three steps:

[0134] a. Wash the bacterial cells obtained by centrifuging the fermentation broth twice with pure water to remove the components of the fermentation broth on the cell surface;

[0135] b. Alkaline lyse the bacterial cells after step a to obtain a cell lysate;

[0136] c. Desalt the lysate after step b to finally obtain a Bifidobacterium longum lysate product.

[0137] Example 4. Analysis of the fermentation product filtrate of Bifidobacterium longum CGMCC No. 31133

[0138] (1)Detection of the main active components of the fermentation product filtrate

[0139] The contents of acetic acid and lactic acid in the fermentation product filtrate were analyzed by high performance liquid chromatography. The liquid phase conditions were as follows: chromatographic column Heshin DK-H 7.8×300 mm, mobile phase: 0.005 mM H 2 SO 4 , flow rate: 0.5 mL / min, column temperature: 40 °C, detector: differential refractive index detector; First, the standard curve equations of acetic acid and lactic acid were prepared as follows: y = 78174487.45 - 1452.27 (R 2 = 0.9988) and y = 81473237.00 + 7595.76 (R 2 = 0.9958);

[0140] The total amino acid content was detected by the ninhydrin method, and the standard curve equation of amino acid was drawn as y = 0.0377x - 0.0146 (R² = 0.9940);

[0141] The protein content was detected by the Coomassie Brilliant Blue method, and the standard curve equation of protein was drawn as y = 6.36x + 0.015 (R 2 = 0.9982);

[0142] The polysaccharide of Bifidobacterium longum was detected by the phenol-sulfuric acid method, and the standard curve equation of total sugar was drawn as y = 13.406x - 0.0253 (R 2 = 0.9950);

[0143] The contents of the main components corresponding to the fermentation product filtrates obtained from Examples 1, 2 and 3 were as follows:

[0144] Table 1 Analysis of the main active components of the fermentation product filtrates of Examples 1, 2 and 3

[0145] Acetic acid (g / L) Lactic acid (g / L) Amino acids (g / L) Protein (g / L) Bifidobacterium longum polysaccharide (g / L) Example 1 18 50 8 5 19 Example 2 17 45 6 1.5 16 Example 3 16 40 3 4.5 18

[0146] (2) Identification of the polysaccharide of Bifidobacterium longum in the fermentation product filtrate

[0147] Take 1 mL of the fermentation product filtrate of Bifidobacterium longum, add an appropriate amount of organic solvent to precipitate the protein, centrifuge to remove the precipitate, then add 3 mL of 95% ethanol for food use to precipitate the polysaccharide, and then filter to obtain the polysaccharide. After vacuum drying, a polysaccharide product is obtained for infrared spectrum analysis. The results are as Figure 1 . The infrared spectrum was measured by the potassium bromide tablet pressing method, and the blank potassium bromide tablet was used as a control group. Scanning was carried out in the range of 4000 - 400 cm −1 with a spectral resolution of 4 cm −1 . As can be seen from Figure 1 , at 3423 cm -1 , there is a stretching vibration peak of the -OH bond. At 2929 cm -1At [specific position], it is the C-H stretching vibration peak, and these two peaks are the characteristic absorption peaks of carbohydrates; 1650 cm -1 At the asymmetric C=O stretching vibration peak, it indicates that the sample has -COO - , indicating the presence of uronic acid in the polysaccharide; 1100 - 1010 cm -1 There are two absorption peaks (1026 cm -1 and 1081 cm -1 ), indicating that the polysaccharide has a furanoside structure. The absorption peak at 856 cm -1 is the characteristic absorption peak of the vibration of sugar molecules, which is the α-type absorption peak of the sugar ring, representing that the polysaccharide is an α-type polysaccharide. Through the structural analysis of this polysaccharide and comparison with the structures of other existing biological polysaccharides, the results show that this polysaccharide is a new type of biological polysaccharide.

[0148] (3)Detection of skin brightening and moisturizing effects of the fermentation product filtrate

[0149] ① Detection of skin brightening effect

[0150] To investigate the skin brightening effect of the filtrate of the Bifidobacterium longum fermentation product of the present invention on tyrosinase activity, 1 mL of the fermentation product filtrate was mixed with 1 mL (100 units) of tyrosinase and incubated at 25 °C for 10 min. Then 1 mL of 0.15% L-DOPA reaction solution was added and incubation continued for 2 min. Immediately, the light absorption value A was measured at 475 nm with a spectrophotometer. The inhibition rate of tyrosinase activity was calculated according to the following formula:

[0151] Inhibition rate (%) = (1 - A 滤液 / A 空白 ) × 100%

[0152] where A 滤液 is the enzyme activity after adding the filtrate, and A 空白 is the blank control without adding the filtrate

[0153] The experimental results show that the tyrosinase inhibition rate of the fermentation product filtrate of the present invention can reach 94.58%, showing excellent skin brightening effect.

[0154] ② Detection of moisturizing effect

[0155] To investigate the moisturizing performance of the filtrate of the fermentation product of the present invention through a water retention experiment with different concentrations of glycerol. 1 mL of the filtrate and different concentrations of glycerol were respectively added into centrifuge tubes and left open. The weight was measured every 3 h. There were three parallel groups. The formula for calculating the water loss rate is as follows:

[0156] Water loss rate = (M 2 - M 3 ) / (M 2 - M1 ) × 100%

[0157] where M 1 is the weight of the centrifuge tube; M 2 is the sum of the weight of the centrifuge tube and the test solution before the experiment; M 3 is the sum of the weight of the centrifuge tube and the test solution at different times

[0158] The experimental results show that the moisturizing performance of the fermentation product filtrate is equivalent to that of 25% glycerol in terms of water retention, indicating that the filtrate has excellent moisturizing performance.

[0159] Example 5. Analysis of the lysate of Bifidobacterium longum CGMCC No. 31133

[0160] (1) Detection of the main active components of the lysate

[0161] By detecting the amino acid content, protein content, and cell wall polysaccharide content of the main active components of the Bifidobacterium longum lysate produced in Examples 1, 2, and 3, the specific contents are shown in Table 2.

[0162] Table 2 Analysis of the main active components of the lysates of Examples 1, 2, and 3

[0163] Wet cell weight (g / L) Amino acids (g / L) Protein (g / L) Cell wall polysaccharide (g / L) Example 1 150 18 19 18 Example 2 135 16 15 16 Example 3 125 15 17 15

[0164] (2) Detection of the performance of the lysate

[0165] ① Detection of antioxidant performance

[0166] Using a 96-well plate, by measuring the absorbance values of different concentrations of Trolox standard solutions at 405 nm, the Trolox standard curve equation was plotted as: y = -1.1661x + 1.5022 (R 2 = 0.9951). Through the free radical scavenging rate experiment of the lysate of the present invention and the lysate products on the market, the experimental results show that the antioxidant capacity of the lysate obtained by the present invention is as high as 92.13%, while the antioxidant capacity of similar products on the market is only 35.43%. Therefore, the lysate obtained by the present invention has higher antioxidant performance.

[0167] ② Detection of anti-aging efficacy

[0168] The anti-aging effect of the lysate was detected by using the zebrafish embryo reactive oxygen species (ROS) scavenging test method. 48-hour-old zebrafish embryos were respectively exposed to the lysate sample solution, and a blank control group was set at the same time. After 24 hours of exposure, the fish embryos were stained with H2DCFDA, and fluorescence photography was used to measure the ROS signal intensity and statistical analysis was carried out. The experimental results show that the lysate of the present invention has a scavenging rate of up to 38% for ROS in zebrafish embryos, can significantly scavenge ROS in zebrafish embryos, and has a significant anti-aging effect.

[0169] ③ Brightening effect detection

[0170] The brightening effect of the filtrate obtained by investigating the inhibition of tyrosinase activity by the lysate of Bifidobacterium longum of the present invention was examined. After diluting 0.1 mL of the lysate to 1 mL with pure water, it was mixed with 1 mL (100 units) of tyrosinase and incubated at 25 °C for 10 min. Then 1 mL of 0.15% L-DOPA reaction solution was added and incubation continued for 2 min. Immediately, the light absorption value A was measured at 475 nm with a spectrophotometer, and the blank without the lysate was used as a control. The experimental results showed that the tyrosinase inhibition rate of the lysate of the present invention could reach 96.25%, indicating an excellent brightening effect.

[0171] ④ Skin repair effect detection

[0172] The skin maintenance and soothing effect of the lysate was tested by the method of inhibiting neutrophil aggregation in zebrafish embryos. The fish embryos were respectively exposed to 10 μM anhydrous copper sulfate and the lysate sample solution, and at the same time, a blank control group, a positive control group and a model control group were set up. After 40 min of exposure, the fish embryos were fixed and stained with Sudan black. The number of neutrophils in the lateral line region was counted and statistically analyzed. The experimental results showed that the neutrophil aggregation inhibition rate of the lysate of the present invention was as high as 95%, which could significantly inhibit neutrophil aggregation in zebrafish embryos and had a significant soothing effect.

Claims

1. A method for preparing a filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof, characterized in that :The production process consists of the following process steps: (1) inoculating Bifidobacterium longum CGMCC No.31133 into a slant culture medium in a test tube to prepare a slant culture; (2) Expanding the culture of the slant strain to serve as seed solution; (3) placing the seed liquid in a fermentation tank for fermentation; (4) preparing a fermentation product filtrate, wherein the fermentation product filtrate contains acetic acid, lactic acid, amino acids, protein, and Bifidobacterium longum polysaccharide; (5) Preparation of Bifidobacterium longum lysate; The slant culture medium of step (1) is composed of the following components in parts by weight: 10-30 parts of glucose, 1-5 parts of tryptone, 1-5 parts of beef peptone, 1-5 parts of yeast extract powder, 1-5 parts of sodium acetate, 1-2 parts of diammonium hydrogen citrate, 1-2 parts of dipotassium hydrogen phosphate, and 15 parts of agar, and water is added to make up to 1000 parts; The seed liquid culture medium of step (2) and the fermentation liquid culture medium of step (3) are composed of the following components in parts by weight: 10-30 parts of glucose, 1-5 parts of tryptone, 1-5 parts of beef peptone, 1-5 parts of yeast extract powder, 1-5 parts of sodium acetate, 1-2 parts of diammonium hydrogen citrate, 1-2 parts of dipotassium hydrogen phosphate and 1000 parts of water; The fermentation process conditions of the fermentation tank are: inoculation amount 10%, fermentation temperature 30-40℃, closed refining, fermentation time 2-8 hours, acetic acid content 5-20g / L, lactic acid content 10-60g / L, amino acid content 0.5-10g / L, protein content 0.1-5g / L, polysaccharide content 0.1-20g / L in the filtrate of the long bifidobacterium fermentation product; the wet weight of the long bifidobacterium somatic cells is 20-150g / L; The bacterial cells are lysed by alkaline lysis to lyse the cell walls; the obtained lysate has an amino acid content of 0.5-20 g / L, a protein content of 0.5-20 g / L and a cell wall polysaccharide content of 0.1-20 g / L, which is a long bifidobacterium lysate product.

2. The method for preparing a filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof according to claim 1, characterized in that: The process conditions for preparing the slant strain are: culture temperature of 30-40°C and culture time of 4-24 hours.

3. The method for preparing a filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof according to claim 1, characterized in that: The seed liquid culture conditions of step (2) are: culture temperature 30-40°C, static closed culture, and culture time 4-18 hours.

4. The method for preparing a filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof according to claim 1, characterized in that: The preparation of the fermentation product filtrate in step (4) comprises the following three steps: a. After fermentation, the fermented liquid is centrifuged to remove bacteria; b. Depigmenting the supernatant after step a; c. Desalt the decolorized solution obtained in step b.

5. The method for preparing a filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof according to claim 1, characterized in that: The preparation of the Bifidobacterium longum lysate in step (5) comprises the following three steps: a. Wash the bacterial cells obtained by centrifugation of the fermentation broth twice with pure water to remove the fermentation broth components on the cell surface; b. Performing alkaline lysis on the bacterial cells after step a to obtain cell lysate; c. Desalt the lysate obtained in step b.

6. A filtrate of a fermentation product of Bifidobacterium longum and a lysate thereof, characterized in that: The method is prepared by the method for preparing the filtrate of the fermentation product of Bifidobacterium longum and its lysate as described in any one of claims 1 to 5.

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