Bifidobacterium lactis BL-99 bacteriocin as well as preparation method and antioxidant application thereof

Abstract

The invention discloses bifidobacterium lactis BL-99 bacteriocin as well as a preparation method and antioxidant application thereof, the bifidobacterium lactis BL-99 bacteriocin comprises exocytosis of the bifidobacterium lactis BL-99 and metabolite of the bifidobacterium lactis BL-99, the mass ratio of citric acid to L-methionine in the bacteriocin is greater than 2: 1, and the preservation number of the bifidobacterium lactis BL-99 is CGMCC (China General Microbiological Culture Collection Center) No.15650. The bifidobacterium lactis BL-99 bacteriocin disclosed by the invention has an antioxidant function, can be applied to foods and medicines, and compared with a mixture comprising complete dead cells, cell wall components, cell membrane components and acellular supernatant, the bacteriocin disclosed by the invention is liquid or water-soluble powder, and has more advantages when being used in some liquid beverages and medicines.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a secretin of Bifidobacterium lactis BL-99, a preparation method thereof and an antioxidant application thereof. Background Art

[0002] At present, postbiotics are attracting much attention as a hot research field. In 2021, the International Scientific Association of Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics. Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to host health. As derivatives of prebiotics and probiotics, postbiotics have multiple potential benefits. They can regulate intestinal flora, enhance intestinal barrier function, regulate intestinal inflammatory responses, etc., thereby having a positive impact on human intestinal health. In addition, the biological activity of postbiotics is not limited to the intestine. It also has biological activities such as inhibiting oral pathogens and regulating lung inflammatory responses. As people's understanding of intestinal health and microbiome continues to deepen, postbiotics are considered to be a potential functional food and health management method, and have received special attention from the industry.

[0003] At present, reports on the bioactivity of postbiotics mainly refer to the bioactivity of probiotics or bacteria. Bifidobacterium lactis BL-99 is a relatively typical postbiotic strain. Previous studies have found that Bifidobacterium lactis BL-99 has potential in regulating intestinal flora, regulating blood pressure and regulating sleep, but the specific form and preparation method of the product will affect its performance.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a secretin of Bifidobacterium lactis BL-99, a preparation method and an antioxidant application thereof, so as to obtain a product with antioxidant properties.

[0006] The Bifidobacterium lactis BL-99 secretin of the present invention refers to a product obtained by extracting water-soluble metabolites of Bifidobacterium lactis BL-99 in bacterial sludge separated from Bifidobacterium lactis BL-99 and exocytosis of Bifidobacterium lactis BL-99.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a secretory agent of Bifidobacterium lactis BL-99, comprising exocytosis products of Bifidobacterium lactis BL-99 and metabolites of Bifidobacterium lactis BL-99, wherein the mass ratio of citric acid to L-methionine in the secretory agent is greater than 2:1, and the preservation number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.

[0009] In the present application, the bacteriocin mainly includes components such as exocytosis and bacterial metabolites. As the bacteriocin is extracted, the concentrations of citric acid and L-methionine in the bacteriocin gradually increase. Therefore, in the present application, the concentrations of L-methionine and citric acid are used to characterize the bacteriocin.

[0010] The bacteriocin of Bifidobacterium lactis BL-99 in the present application has antioxidant function and can be used in food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin in the present application is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

[0011] In an optional embodiment, the mass ratio of citric acid to L-methionine in the bacteriocin is 2-25:1, specifically, it can be 2:1, 5:1, 10:1, 15:1, 20:1, 25:1 or any value between 2-25:1, or any value greater than 25:1.

[0012] Preferably, the mass ratio of citric acid to L-methionine in the bacteriocin is 8-9:1.

[0013] In an optional embodiment, the concentration of L-methionine in the bacteriocin is 0.2-1 mg / ml, and the concentration of citric acid is 2-5 mg / ml.

[0014] Specifically, the separation of the bacterial sludge and the supernatant in the present application can adopt an existing separation method, for example, centrifugation can be selected to achieve solid-liquid separation; in some embodiments, the concentration of L-methionine in the secretory agent is 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.51 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml or any value between 0.2-1 mg / ml, or any value greater than 1 mg / ml; the concentration of citric acid is 2 mg / ml, 3 mg / ml, 5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml or any value between 2-5 mg / ml, or any value greater than 5 mg / ml.

[0015] Preferably, the concentration of L-methionine in the bacteriocin is 0.25-0.3 mg / ml, and the concentration of citric acid is 2-2.8 mg / ml.

[0016] In a second aspect, the present application provides a method for producing the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:

[0017] Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

[0018] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

[0019] In an optional embodiment, the extraction temperature is 0-37°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C or any value between 0-37°C; preferably, the extraction temperature is 3-5°C.

[0020] In an optional embodiment, the extraction time is 0.5-3 h, specifically 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h or any value between 1-3 h; preferably, the extraction time is 30-90 min.

[0021] In an optional embodiment, the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 0.5×10 11 -5×10 11 , specifically 0.5×10 11 cfu / mL, 0.7×10 11 cfu / mL, 0.9×10 11 cfu / mL, 1×10 11 cfu / mL, 2×10 11 cfu / mL, 3×10 11 cfu / mL, 4×10 11 cfu / mL, 5×10 11 cfu / mL or 0.5×10 11 -5×10 11 cfu / mL; preferably, the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

[0022] In an optional embodiment, the temperature of heat sterilization is 70-121°C, specifically 70°C, 80°C, 90°C, 100°C, 110°C, 121°C or any value between 70-121°C; preferably, the temperature of heat sterilization is 70-100°C.

[0023] In an optional embodiment, the heat sterilization time is 5-30 min, specifically 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value between 5-30 min; preferably, the heat sterilization time is 10-16 min.

[0024] In an optional embodiment, the method further comprises the step of fermentation: culturing Bifidobacterium lactis BL-99 in liquid culture, and stopping the fermentation when the Bifidobacterium lactis BL-99 grows to the logarithmic phase;

[0025] Preferably, the fermentation step comprises: inoculating Bifidobacterium lactis BL-99 fermentation seed liquid into a culture medium, and first fermenting for 14-16 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.8-6.2 to obtain Bifidobacterium lactis BL-99 fermentation liquid; preferably, the preparation of the Bifidobacterium lactis BL-99 fermentation seed liquid comprises: activating the Bifidobacterium lactis BL-99 strain, purifying it, culturing it with a first-level seed liquid, expanding it with a second-level seed liquid, culturing it with a third-level seed liquid, and preparing fermentation seeds to obtain the Bifidobacterium lactis BL-99 fermentation seed liquid;

[0026] Preferably, the culture medium is MRS liquid culture medium.

[0027] In an optional embodiment, the solvent is water; preferably, the solvent is sterile water, specifically sterile purified water;

[0028] Preferably, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

[0029] In an optional embodiment, the concentration of L-methionine in the bacteriocin is 0.2-1 mg / ml, and the concentration of citric acid is 2-5 mg / ml.

[0030] Specifically, the separation of the bacterial sludge and the supernatant in the present application can adopt an existing separation method, for example, centrifugation can be selected to achieve solid-liquid separation; in some embodiments, the concentration of L-methionine in the secretory agent is 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.51 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml or any value between 0.2-1 mg / ml, or any value greater than 1 mg / ml; the concentration of citric acid is 2 mg / ml, 3 mg / ml, 5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml or any value between 2-5 mg / ml, or any value greater than 5 mg / ml.

[0031] Preferably, the concentration of L-methionine in the bacteriocin is 0.25-0.3 mg / ml, and the concentration of citric acid is 2-2.8 mg / ml.

[0032] After the sterilization step, the bacteriocin is freeze-dried to obtain a freeze-dried bacteriocin, in which the solvent is removed, and the mass ratio of citric acid to L-methionine in the freeze-dried bacteriocin is greater than 2:1.

[0033] Specifically, in some embodiments, the secretin of Bifidobacterium lactis BL-99 is prepared by the following steps:

[0034] 1. Preparation of Tertiary Seeds

[0035] 1.1 Standard cryopreservation tubes

[0036] Prepare uniformly from the purified strains, divide into 1.5mL centrifuge tubes, no less than 50 tubes, and store in a -80℃ refrigerator with a shelf life of no more than 6 months.

[0037] 1.2 Activation of cryotubes

[0038] Take a portion of the bacterial strain stored at -80℃, thaw it at room temperature, aseptically take 200μL of bacterial liquid and inoculate it into 10mL of seed liquid culture medium, and culture it at 37℃ for 11-13h.

[0039] 1.3 Primary purification

[0040] Take the cultured bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 11-13h.

[0041] 1.4 Secondary purification

[0042] Take the cultured purified bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 11-13h.

[0043] 1.5 Primary seed preparation

[0044] Select one tube of secondary purified bacterial culture and pipette 200 μL of the bacterial culture into five tubes of 10 mL MRS liquid culture medium. Incubate at 37°C for 11-13 hours.

[0045] 1.6 Secondary seed preparation

[0046] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 11-13 hours.

[0047] 1.7 Preparation of tertiary seeds

[0048] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0049] 1.8 Temporary storage of third-level seeds

[0050] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0051] 1.9 Process Quality Control

[0052] (1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

[0053] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, bacilli-shaped, not in chains, and slightly curved.

[0054] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0055] 2. Fermentation Seed Preparation

[0056] 2.1 Vaccination

[0057] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0058] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermenter to maintain positive pressure in the fermenter for 5-10 minutes.

[0059] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0060] (4) Unscrew the top of the inoculator and pour the cultured seeds into the sterile area above the flame, with an inoculation rate of 2.5%.

[0061] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0062] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0063] 2.2 Fermentation

[0064] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0065] (2) The fermentation time was 11 h to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0066] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0067] 2.3 Process quality control

[0068] (1) Seed tank fermentation endpoint determination indicators: pH 4.2-4.6, OD600 ≥ 2.0.

[0069] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, in the form of rods, not in chains, slightly curved, and in an arc shape.

[0070] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0071] 3. Fermentation Broth Culture

[0072] 3.1 Vaccination

[0073] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0074] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0075] (3) Open the nitrogen inlet valve and fill with nitrogen for 5-10 minutes. Open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation. The inoculation amount is 2%.

[0076] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0077] (5) Close the nitrogen inlet valve and the discharge valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0078] 3.2 Fermentation

[0079] (1) Set the fermentation parameters to 30-40°C, 60-80 rpm, and a constant pH of 5.8-6.2.

[0080] (2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0081] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

[0082] 3.3 Mushroom sludge separation

[0083] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0084] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0085] (3) Open the feed valve, and the fermentation liquid of Bifidobacterium lactis BL-99 enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0086] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0087] 4. Bacteriocin Extraction

[0088] 4.1 Mud transfer

[0089] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0090] 4.2 Extraction conditions

[0091] The bacterial mud was extracted and sterile purified water was used to mix the bacterial mud to obtain a mixed solution. The total colony count of Bifidobacterium lactis BL-99 in the mixed solution was 0.5×10 11 -5×10 11 cfu / mL, extraction temperature 0-37°C, and extraction time 0.5-3h.

[0092] It should be noted that in the actual production process, the OD600 value of the Lactobacillus paracasei K56 fermented liquid in the 3.2 step can be adjusted, and the add-on of sterile purified water in the control extraction step can be simultaneously controlled so that the volume of the mixed liquor is 1% of the volume of the Lactobacillus paracasei K56 fermented liquid, so as to more easily adjust the total colony count of Lactobacillus paracasei K56 in the mixed liquor.

[0093] 5. Centrifugal separation

[0094] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0095] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0096] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0097] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0098] (5) Transfer the centrifuged supernatant to a collection tank.

[0099] 6. Packaging and sterilization

[0100] The filled bottled supernatant was heat sterilized to obtain the bacteriocin. The sterilization conditions for each strain were as follows:

[0101] The sterilization conditions of Bifidobacterium lactis BL-99 are 70-121°C for 5-30 minutes.

[0102] In a third aspect, the present invention provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in preparing a food, wherein the food comprises at least one of a dairy product and a beverage.

[0103] In a fourth aspect, the present invention provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in preparing a composition, wherein the composition comprises an antioxidant composition; preferably, the composition is selected from at least one of a medicine, a health food and a feed.

[0104] The present invention has the following beneficial effects:

[0105] The bacteriocin of Bifidobacterium lactis BL-99 in the present application has antioxidant function and can be used in food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin in the present application is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0107] Figure 1 This is a flow chart of the preparation of secretory agents from Bifidobacterium lactis BL-99 in Example 1;

[0108] Figure 2 This is a physical picture of the secretory activity of Bifidobacterium lactis BL-99 in Example 1;

[0109] Figure 3 Example 1 obtained a liquid chromatogram of a bacteriocin sample;

[0110] Figure 4 is the hydroxyl radical scavenging ability of secretin of Bifidobacterium lactis BL-99 under different extraction conditions;

[0111] Figure 5 is the DPPH free radical scavenging ability of secretin of Bifidobacterium lactis BL-99 under different extraction conditions;

[0112] Figure 6 It is the hydroxyl radical scavenging ability and DPPH free radical scavenging ability of the inactivated bacteria in Comparative Example 1 at different heat sterilization temperatures. DETAILED DESCRIPTION

[0113] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0114] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0115] Example 1

[0116] This embodiment provides a kind of Bifidobacterium lactis BL-99 secretory factor, such as Figure 1 As shown, the preparation method comprises the following steps:

[0117] 1. Preparation of Tertiary Seeds

[0118] 1.1 Standard cryopreservation tubes

[0119] Prepare uniformly from the purified strains, divide into 1.5mL centrifuge tubes, no less than 50 tubes, and store in a -80℃ refrigerator with a shelf life of no more than 6 months.

[0120] 1.2 Activation of cryotubes

[0121] Take a portion of the bacterial strain stored at -80℃, thaw it at room temperature, aseptically take 200μL of bacterial liquid and inoculate it into 10mL of seed liquid culture medium, and culture it at 37℃ for 11-13h.

[0122] 1.3 Primary purification

[0123] Take the cultured bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 11-13h.

[0124] 1.4 Secondary purification

[0125] Take the cultured purified bacterial liquid for dilution and coating, with dilutions of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h-72h until obvious colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 11-13h.

[0126] 1.5 Primary seed preparation

[0127] Select one tube of secondary purified bacterial culture and pipette 200 μL of the bacterial culture into five tubes of 10 mL MRS liquid culture medium. Incubate at 37°C for 11-13 hours.

[0128] 1.6 Secondary seed preparation

[0129] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 11-13 hours.

[0130] 1.7 Preparation of tertiary seeds

[0131] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0132] 1.8 Temporary storage of third-level seeds

[0133] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0134] 1.9 Process Quality Control

[0135] (1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

[0136] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, bacilli-shaped, not in chains, and slightly curved.

[0137] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0138] Formula of three-stage seed fermentation medium

[0139]

[0140] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min

[0141] 2. Fermentation Seed Preparation

[0142] 2.1 Vaccination

[0143] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0144] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermenter to maintain positive pressure in the fermenter for 5-10 minutes.

[0145] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0146] (4) Unscrew the top of the inoculator and pour the cultured seeds into the sterile area above the flame, with an inoculation rate of 2.5%.

[0147] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0148] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0149] 2.2 Fermentation

[0150] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0151] (2) The fermentation time was 11 h to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0152] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0153] 2.3 Process quality control

[0154] (1) Seed tank fermentation endpoint determination indicators: pH 4.2-4.6, OD600 ≥ 2.0.

[0155] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, in the form of rods, not in chains, slightly curved, and in an arc shape.

[0156] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0157] Seed tank fermentation medium formula

[0158]

[0159] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0160] 3. Fermentation Broth Culture

[0161] 3.1 Vaccination

[0162] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0163] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0164] (3) Open the nitrogen inlet valve and fill with nitrogen for 5-10 minutes. Open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation. The inoculation amount is 2%.

[0165] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0166] (5) Close the nitrogen inlet valve and the discharge valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0167] 3.2 Fermentation

[0168] (1) Set the fermentation parameters to 37°C, 70 rpm, and a constant pH of 6.

[0169] (2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0170] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

[0171] Fermentation medium formula for fermentation tank

[0172]

[0173] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0174] 3.3 Mushroom sludge separation

[0175] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0176] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0177] (3) Open the feed valve, and the fermentation liquid of Bifidobacterium lactis BL-99 enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0178] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0179] 4. Bacteriocin Extraction

[0180] 4.1 Mud transfer

[0181] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0182] 4.2 Extraction conditions

[0183] The bacterial mud was extracted and sterile purified water was used to mix the bacterial mud to obtain a mixed solution. The total colony count of Bifidobacterium lactis BL-99 in the mixed solution was 1×1011 cfu / mL, 1.5×10 11 cfu / mL, 3×10 11 cfu / mL, the extraction temperatures were 4°C, 25°C, and 37°C, the extraction times were 1h, 2h, and 3h, and the extraction speed was 70rpm.

[0184] 5. Centrifugal separation

[0185] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8-2.5 bar.

[0186] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0187] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0188] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0189] (5) Transfer the centrifuged supernatant to a collection tank.

[0190] 6. Packaging and sterilization

[0191] The filled bottled supernatant is heat sterilized to obtain the secretory agent. The sterilization conditions of Bifidobacterium lactis BL-99 are 75°C for 10 min. The secretory agent of Bifidobacterium lactis BL-99 is as follows: Figure 2 shown.

[0192] 7. Quantitative Detection

[0193] The concentrations of L-methionine and citric acid in the product obtained in Example 1 were detected, and the detection method comprised the following steps:

[0194] (1) Preparation of standard working solution

[0195] Standard stock solution: Accurately weigh appropriate amounts of standard substances (i.e., L-methionine and citric acid, accurate to 0.1 mg), dissolve in water and prepare standard stock solutions with a concentration of 5 mg / mL, and store at -20°C.

[0196] Mixed standard intermediate solution: Accurately pipette appropriate volumes of standard stock solutions, dilute to volume with water, prepare a mixed standard intermediate solution with a concentration of 500 μg / mL, and store at 4°C.

[0197] Mixed standard working solution: dilute the mixed standard intermediate solution step by step with water as needed to prepare mixed standard working solutions with concentrations of 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively. Prepare them before use.

[0198] (2) Preparation of elution solution

[0199] 0.1% phosphoric acid aqueous solution: Take 1 mL of phosphoric acid, dilute it with water and make up to 1000 mL, mix well, and use it immediately.

[0200] 0.1% phosphoric acid acetonitrile solution: Take 1 mL of phosphoric acid, dilute with acetonitrile and make up to 1000 mL, mix well, and use immediately.

[0201] (3) Preparation of test solution

[0202] Liquid sample: Mix the secretory sample of Bifidobacterium lactis BL-99 and directly aspirate 1 mL. Centrifuge at 10,000 rpm for 10 min at 4°C. Dilute the supernatant to the linear range and load it onto the column for analysis.

[0203] (4) Detection and analysis

[0204] The sample solution and the standard working solution were tested by high performance liquid chromatography, and the corresponding chromatogram peak area was determined. The standard curve was drawn with the concentration of the standard working solution as the horizontal axis and the peak area of the chromatogram as the vertical axis. Figure 3 , where 1 is L-methionine and 2 is citric acid), the concentrations of L-methionine and citric acid in the bacteriocin extraction step were calculated based on the standard curve.

[0205] Chromatographic analysis conditions: An LC-20A analytical system was used, with a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A was 0.1% phosphoric acid in water; mobile phase B was 0.1% phosphoric acid in acetonitrile; the gradient elution program is shown in Table 1; the flow rate was 0.7 mL / min; the detection wavelength was 210 nm; the column temperature was 30°C; and the injection volume was 5 μL.

[0206] Table 1 Gradient elution program

[0207] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 33 95 5 45 95 5

[0208] The total number of Bifidobacterium lactis BL-99 colonies in the mixed solution was calculated to be 3×10 11 cfu / mL, the extraction temperature was 4℃, and the extraction time was 1h. The L-methionine concentration in the bacteriocin was 0.29mg / 100ml and the citric acid concentration was 2.48mg / 100ml.

[0209] (5) Calculate the number of colonies before inactivation of inactivated bacteria

[0210] Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

[0211] When citric acid was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 2.289x-4.313; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0212] When L-methionine was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 18.702x + 4.464; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0213]

[0214] Comparative Example 1

[0215] This embodiment provides a fermentation-inactivated bacterium of Bifidobacterium lactis BL-99, the preparation method of which comprises the following steps:

[0216] 1. Preparation of tertiary seeds is the same as in Example 1.

[0217] 2. Preparation of fermented seeds, same as in Example 1.

[0218] 3. Fermentation broth culture, same as Example 1.

[0219] 4. Bacteria inactivation

[0220] Sterile purified water was mixed with the bacterial sludge and resuspended to obtain a mixed solution (the concentration was the same as in Example 1), and the mixed solution was heat sterilized at temperatures of 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min.

[0221] Test Example 1: 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging

[0222] 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of mixed solution (10 9cfu / mL) as raw material for the bacteriocin or inactivated bacteria and incubate at 25°C in the dark for 30 minutes. The control group was replaced with an equal volume of PBS (pH 7.4), while the blank group was replaced with an equal volume of PBS (pH 7.4) instead of the DPPH free radical solution. After centrifugation at 2,000 × g for 10 minutes, the absorbance of the solution was measured at 517 nm. The calculation formula is as follows:

[0223]

[0224] Test Example 2: Hydroxyl Radical Scavenging

[0225] A total of 1.0 mL of the mixture (10 9 cfu / mL) as the starting material for the bacteriocin preparation or inactivated bacteria. After adding 20 mM H2O2 (1.0 mL), the mixture was incubated in a 37°C water bath for 90 min. The absorbance was measured at 536 nm. The hydroxyl radical scavenging activity was calculated as follows:

[0226]

[0227] In this application, LC-MS was used to detect the components of the inactivated bacteria obtained in Comparative Example 1, and the peak areas greater than 10 4 The components of the bacteriocin were screened to obtain targets that characterize the inactivated bacteria components. The target screening criteria include: ① it does not exist before heat sterilization, or the content of the component before heat sterilization is much lower than the content of the component after heat sterilization; ② within the experimental range, it can stably exist under different heat sterilization conditions (content change range ≤ 20%). It was found that 6 substances can be used as potential detection targets, namely: Proly-Alanine, L-Methioniine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic peptide, GRPPK. At the same time, the 6 detection targets in bacteriocin were detected, and it was found that only citric acid and L-methionine were present in bacteriocin, and the other four detection targets were not extracted or the content was below the detection line. Therefore, in this application, citric acid and L-methionine are used as detection targets to characterize bacteriocin.

[0228] The test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 1 are as follows: Figure 4 As shown, Figure 4 It can be seen that when the extraction concentration is 3×10 11cfu / mL, the extraction temperature was 4 ° C, and the extraction time was 1 h, the hydroxyl radical scavenging ability of the bacteriocin was better; the test results of the DPPH radical scavenging ability of the bacteriocin obtained in Example 1 are as follows Figure 5 As shown, Figure 5 The extraction temperature and time of K1-K9 were 4℃, 1h, 4℃, 2h, 4℃, 3h, 25℃, 1h, 25℃, 2h, 25℃, 3h, 37℃, 1h, 37℃, 2h and 37℃, 3h respectively; the extraction temperature and time of K10-K18 were 4℃, 1h, 4℃, 2h, 4℃, 3h, 25℃, 1h, 25℃, 2h, 25℃, 3h, 37℃, 1h, 37℃, 2h and 37℃, 3h respectively; the extraction temperature and time of K19-K27 were 4℃, 1h, 4℃, 2h, 4℃, 3h, 25℃, 1h, 25℃, 2h, 25℃, 3h, 37℃, 1h, 37℃, 2h and 37℃, 3h respectively. Figure 5 The extraction conditions had relatively little effect on DPPH free radicals.

[0229] The hydroxyl radical scavenging ability and DPPH radical scavenging ability of the postbiotics obtained in Comparative Example 1 are as follows: Figure 6 As shown in the figure, the DPPH radical scavenging ability of mycobacterin did not change much with temperature at first, but it decreased significantly under high temperature conditions of 121℃.

[0230] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A secretin of Bifidobacterium lactis BL-99, characterized in that The invention comprises exocytosis product of Bifidobacterium lactis BL-99 and metabolites of Bifidobacterium lactis BL-99. The mass ratio of citric acid to L-methionine in the secretory substance is greater than 2:

1. The preservation number of Bifidobacterium lactis BL-99 is CGMCC No.15650.

2. The Bifidobacterium lactis BL-99 secretin according to claim 1, characterized in that The mass ratio of citric acid to L-methionine in the bacteriocin is 2-25:1; Preferably, the mass ratio of citric acid to L-methionine in the bacteriocin is 8-9:

1.

3. A method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 1 or 2, characterized in that: The following steps are involved: Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid; Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

4. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 3, wherein The extraction temperature is 0-37°C; preferably, the extraction temperature is 3-5°C; And / or, the extraction time is 0.5-3h; preferably, the extraction time is 30-90min.

5. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 3, wherein The total colony count of Bifidobacterium lactis BL-99 in the mixed solution was 0.5×10 11 -5×10 11 cfu / mL; preferably, the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

6. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 3, wherein: The temperature for heat sterilization is 70-121°C; preferably, the temperature for heat sterilization is 70-100°C.

7. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 3, wherein: The heat sterilization time is 5-30 minutes; preferably, the heat sterilization time is 10-16 minutes.

8. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 3, wherein: The invention also includes a fermentation step of culturing Bifidobacterium lactis BL-99 in liquid culture and stopping the fermentation when the Bifidobacterium lactis BL-99 grows to the logarithmic phase; Preferably, the fermentation step comprises: inoculating Bifidobacterium lactis BL-99 fermentation seed liquid into a culture medium, and first fermenting for 14-16 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.8-6.2 to obtain Bifidobacterium lactis BL-99 fermentation liquid; preferably, the preparation of the Bifidobacterium lactis BL-99 fermentation seed liquid comprises: activating the Bifidobacterium lactis BL-99 strain, purifying it, culturing it with a first-level seed liquid, expanding it with a second-level seed liquid, culturing it with a third-level seed liquid, and preparing fermentation seeds to obtain the Bifidobacterium lactis BL-99 fermentation seed liquid; Preferably, the culture medium is MRS liquid culture medium; Preferably, the solvent is water; preferably, the solvent is sterile water; Preferably, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

9. Use of the secretin of Bifidobacterium lactis BL-99 according to claim 1 or 2 in preparing food, characterized in that: The food comprises at least one of dairy products and beverages.

10. Use of the secretin of Bifidobacterium lactis BL-99 according to claim 1 or 2 in preparing a composition, characterized in that: The composition includes an antioxidant composition; preferably, the composition is selected from at least one of medicines, health foods and feeds.

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