Bifidobacterium lactis bl-99 bacteriocin, inactivated bacteria, preparation method, and use thereof

Abstract

Disclosed are Bifidobacterium lactis BL-99 bacteriocin, inactivated bacteria, a preparation method, and a use thereof. The Bifidobacterium lactis BL-99 bacteriocin comprises the exocytotic substances and metabolic products of 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 No.15650. The Bifidobacterium lactis BL-99 bacteriocin has antioxidant functions and can be applied in food and pharmaceuticals. Moreover, compared with mixtures comprising whole dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin is a liquid or water-soluble powder, and offers additional advantages for use in certain liquid beverages and pharmaceuticals.

Description

Bifidobacterium lactis BL-99 secretory factor, inactivated bacteria, preparation method and application thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims priority to Chinese patent application number 202410174812.1 filed with the Patent Office of China on February 7, 2024, entitled “Bifidobacterium lactis BL-99 secretin, preparation method and antioxidant application thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of microbial technology, and in particular to a secretin of Bifidobacterium lactis BL-99, an inactivated bacterium, a preparation method and applications thereof. Background Art

[0004] 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.

[0005] Currently, reports on the bioactivity of postbiotics primarily refer to the bioactivity of probiotics or bacteria. Bifidobacterium lactis BL-99 is a typical postbiotic strain. Previous studies have found that Bifidobacterium lactis BL-99 has potential in regulating intestinal flora, blood pressure, and sleep. For example, CN201811161053.6 discloses that Bifidobacterium lactis BL-99 has the effect of regulating gastrointestinal flora. However, the specific form and preparation method of the product will affect its performance.

[0006] In view of this, the present disclosure is proposed. Summary of the Invention

[0007] The purpose of the present disclosure is to provide a secretin of Bifidobacterium lactis BL-99, an inactivated bacterium, a preparation method and an application thereof, so as to obtain a product with antioxidant properties.

[0008] The Bifidobacterium lactis BL-99 secretin disclosed in the present invention refers to a product obtained by extracting water-soluble metabolites of Bifidobacterium lactis BL-99 in bacterial sludge isolated from Bifidobacterium lactis BL-99 and exocytosis of Bifidobacterium lactis BL-99.

[0009] The present disclosure is achieved as follows:

[0010] In a first aspect, the present disclosure 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 deposit number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.

[0011] In a second aspect, the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:

[0012] Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

[0013] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

[0014] In a third aspect, the present disclosure provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in preparing food, wherein the food comprises at least one of a dairy product and a beverage.

[0015] In a fourth aspect, the present disclosure 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.

[0016] In a fifth aspect, the present disclosure provides a method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99, comprising resuspending bacterial sludge separated from fermentation broth with a solvent to obtain a mixed solution, and heat sterilizing the mixed solution to obtain the fermented inactivated bacteria of Bifidobacterium lactis BL-99.

[0017] In a sixth aspect, the present disclosure provides a fermented inactivated bacterium of Bifidobacterium lactis BL-99, which is prepared by the method described in any one of the aforementioned embodiments.

[0018] In a seventh aspect, the present disclosure provides a Bifidobacterium lactis BL-99 postbiotic, comprising at least one of the Bifidobacterium lactis BL-99 secretin according to any one of the aforementioned embodiments and the Bifidobacterium lactis BL-99 fermentation-inactivated bacteria according to the aforementioned embodiments.

[0019] In an eighth aspect, the present disclosure provides a use of the Bifidobacterium lactis BL-99 postbiotic 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.

[0020] In a ninth aspect, the present disclosure provides a use of the Bifidobacterium lactis BL-99 postbiotic 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.

[0021] In a tenth aspect, the present disclosure provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in anti-oxidation.

[0022] The present disclosure has the following beneficial effects:

[0023] The bacteriocin of Bifidobacterium lactis BL-99 disclosed in the present invention 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 disclosed in the present invention is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, 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 disclosure 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 creative work.

[0025] FIG1 is a flow chart of the preparation of secretory agents from Bifidobacterium lactis BL-99 in Example 1;

[0026] FIG2 is a physical image of the secretory activity of Bifidobacterium lactis BL-99 in Example 1;

[0027] FIG3 is a liquid chromatogram of a bacteriocin sample obtained in Example 1;

[0028] FIG4 shows the hydroxyl radical scavenging ability of secretin from Bifidobacterium lactis BL-99 under different extraction conditions;

[0029] Figure 5 shows the DPPH radical scavenging ability of secretory agents of Bifidobacterium lactis BL-99 under different extraction conditions;

[0030] FIG6 shows the hydroxyl radical scavenging ability and DPPH radical scavenging ability of the inactivated bacteria in Comparative Example 1 at different heat sterilization temperatures. DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0032] In a first aspect, the present disclosure 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 deposit number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.

[0033] In the present disclosure, bacteriocin mainly includes components such as exocytosis products and bacterial metabolites. As the extraction of bacteriocin proceeds, the concentrations of citric acid and L-methionine in the bacteriocin gradually increase. Therefore, in the present disclosure, the concentrations of L-methionine and citric acid are used to characterize the bacteriocin.

[0034] The bacteriocin of Bifidobacterium lactis BL-99 disclosed in the present invention 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 disclosed in the present invention is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

[0035] 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.

[0036] Optionally, the mass ratio of citric acid to L-methionine in the bacteriocin is 8-9:1.

[0037] 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.

[0038] Specifically, the separation of the bacterial sludge and the supernatant in the present disclosure can adopt existing separation methods, such as selecting a centrifugal method to achieve solid-liquid separation; in some embodiments, the L-methionine concentration in the bacteriocin 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 citric acid concentration 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.

[0039] Optionally, 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.

[0040] In a second aspect, the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:

[0041] Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

[0042] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

[0043] 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; optionally, the extraction temperature is 3-5°C.

[0044] 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; optionally, the extraction time is 30-90 min.

[0045] 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 11cfu / mL, 5×10 11 cfu / mL or 0.5×10 11 -5×10 11 cfu / mL; optionally, the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 2×10 11 -4×10 11 cfu / mL.

[0046] 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; optionally, the temperature of heat sterilization is 70-100°C.

[0047] 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; optionally, the heat sterilization time is 10-16 min.

[0048] 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;

[0049] Optionally, 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; Optionally, 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 seed liquid, expanding it with a second seed liquid, culturing it with a third seed liquid, and preparing fermentation seeds to obtain the Bifidobacterium lactis BL-99 fermentation seed liquid;

[0050] Optionally, the culture medium is MRS liquid culture medium.

[0051] In an optional embodiment, the solvent is water; optionally, the solvent is sterile water, specifically sterile purified water;

[0052] Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

[0053] 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.

[0054] Specifically, the separation of the bacterial sludge and the supernatant in the present disclosure can adopt existing separation methods, such as selecting a centrifugal method to achieve solid-liquid separation; in some embodiments, the L-methionine concentration in the bacteriocin 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 citric acid concentration 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.

[0055] Optionally, 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.

[0056] 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.

[0057] Specifically, in some embodiments, the secretin of Bifidobacterium lactis BL-99 is prepared by the following steps:

[0058] 1. Preparation of Tertiary Seeds

[0059] 1.1 Standard cryopreservation tubes

[0060] 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.

[0061] 1.2 Activation of cryotubes

[0062] 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.

[0063] 1.3 Primary purification

[0064] 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.

[0065] 1.4 Secondary purification

[0066] 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.

[0067] 1.5 Primary seed preparation

[0068] 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.

[0069] 1.6 Secondary seed preparation

[0070] 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.

[0071] 1.7 Preparation of tertiary seeds

[0072] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0073] 1.8 Temporary storage of third-level seeds

[0074] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0075] 1.9 Process Quality Control

[0076] (1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

[0077] (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.

[0078] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0079] 2. Fermentation Seed Preparation

[0080] 2.1 Vaccination

[0081] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0082] (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.

[0083] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0084] (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%.

[0085] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0086] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0087] 2.2 Fermentation

[0088] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0089] (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.

[0090] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0091] 2.3 Process quality control

[0092] (1) Seed tank fermentation endpoint determination indicators: pH 4.2-4.6, OD600 ≥ 2.0.

[0093] (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.

[0094] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0095] 3. Fermentation Broth Culture

[0096] 3.1 Vaccination

[0097] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0098] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0099] (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%.

[0100] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0101] (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.

[0102] 3.2 Fermentation

[0103] (1) Set the fermentation parameters to 30-40°C, 60-80 rpm, and a constant pH of 5.8-6.2.

[0104] (2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0105] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

[0106] 3.3 Mushroom sludge separation

[0107] (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.

[0108] (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.

[0109] (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.

[0110] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0111] 4. Bacteriocin Extraction

[0112] 4.1 Mud transfer

[0113] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0114] 4.2 Extraction conditions

[0115] 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.

[0116] 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.

[0117] 5. Centrifugal separation

[0118] (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.

[0119] (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.

[0120] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0121] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0122] (5) Transfer the centrifuged supernatant to a collection tank.

[0123] 6. Packaging and sterilization

[0124] The filled bottled supernatant was heat sterilized to obtain the bacteriocin. The sterilization conditions for each strain were as follows:

[0125] The sterilization conditions of Bifidobacterium lactis BL-99 are 70-121°C for 5-30 minutes.

[0126] In a third aspect, the present disclosure provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in preparing food, wherein the food comprises at least one of a dairy product and a beverage.

[0127] In a fourth aspect, the present disclosure 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.

[0128] In a fifth aspect, the present disclosure provides a method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99, comprising resuspending bacterial sludge separated from fermentation broth with a solvent to obtain a mixed solution, and heat sterilizing the mixed solution to obtain the fermented inactivated bacteria of Bifidobacterium lactis BL-99.

[0129] In an optional embodiment, the heat sterilization temperature is 70-121°C, specifically 70°C, 80°C, 90°C, 100°C, 110°C, 121°C or any value between 70-121°C; the heat sterilization time is 5-30min, specifically 5min, 10min, 15min, 20min, 25min, 30min or any value between 5-30min.

[0130] 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 cfu / mL, 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 Any value between cfu / mL.

[0131] In a sixth aspect, the present disclosure provides a fermented inactivated bacterium of Bifidobacterium lactis BL-99, which is prepared by the method described in any one of the aforementioned embodiments.

[0132] In a seventh aspect, the present disclosure provides a Bifidobacterium lactis BL-99 postbiotic, comprising at least one of the Bifidobacterium lactis BL-99 secretin according to any one of the aforementioned embodiments and the Bifidobacterium lactis BL-99 fermentation-inactivated bacteria according to the aforementioned embodiments.

[0133] In an eighth aspect, the present disclosure provides a use of the Bifidobacterium lactis BL-99 postbiotic 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.

[0134] In a ninth aspect, the present disclosure provides a use of the Bifidobacterium lactis BL-99 postbiotic 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.

[0135] In a tenth aspect, the present disclosure provides a use of the secretin of Bifidobacterium lactis BL-99 according to any one of the aforementioned embodiments in anti-oxidation.

[0136] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0137] Example 1

[0138] This embodiment provides a secretin of Bifidobacterium lactis BL-99, as shown in FIG1 , and its preparation method comprises the following steps:

[0139] 1. Preparation of Tertiary Seeds

[0140] 1.1 Standard cryopreservation tubes

[0141] 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.

[0142] 1.2 Activation of cryotubes

[0143] 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.

[0144] 1.3 Primary purification

[0145] 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.

[0146] 1.4 Secondary purification

[0147] 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.

[0148] 1.5 Primary seed preparation

[0149] 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.

[0150] 1.6 Secondary seed preparation

[0151] 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.

[0152] 1.7 Preparation of tertiary seeds

[0153] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0154] 1.8 Temporary storage of third-level seeds

[0155] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0156] 1.9 Process Quality Control

[0157] (1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

[0158] (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, slightly curved, and arc-shaped.

[0159] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0160] Formula of three-stage seed fermentation medium

[0161] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min

[0162] 2. Fermentation Seed Preparation

[0163] 2.1 Vaccination

[0164] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0165] (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.

[0166] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0167] (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%.

[0168] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0169] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0170] 2.2 Fermentation

[0171] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0172] (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.

[0173] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0174] 2.3 Process quality control

[0175] (1) Seed tank fermentation endpoint determination indicators: pH 4.2-4.6, OD600 ≥ 2.0.

[0176] (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.

[0177] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0178] Seed tank fermentation medium formula

[0179] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0180] 3. Fermentation Broth Culture

[0181] 3.1 Vaccination

[0182] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0183] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0184] (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%.

[0185] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0186] (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.

[0187] 3.2 Fermentation

[0188] (1) Set the fermentation parameters to 37°C, 70 rpm, and a constant pH of 6.

[0189] (2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0190] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

[0191] Fermentation medium formula for fermentation tank

[0192] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0193] 3.3 Mushroom sludge separation

[0194] (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.

[0195] (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.

[0196] (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.

[0197] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0198] 4. Bacteriocin Extraction

[0199] 4.1 Mud transfer

[0200] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0201] 4.2 Extraction conditions

[0202] 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 1×10 11 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.

[0203] 5. Centrifugal separation

[0204] (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.

[0205] (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.

[0206] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0207] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0208] (5) Transfer the centrifuged supernatant to a collection tank.

[0209] 6. Packaging and sterilization

[0210] The filled bottled supernatant was heat sterilized to obtain the secretory agent. The sterilization conditions of Bifidobacterium lactis BL-99 were 75° C. for 10 min. The obtained secretory agent of Bifidobacterium lactis BL-99 was shown in FIG2 .

[0211] 7. Quantitative Detection

[0212] 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:

[0213] (1) Preparation of standard working solution

[0214] 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.

[0215] 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.

[0216] 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.

[0217] (2) Preparation of elution solution

[0218] 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.

[0219] 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.

[0220] (3) Preparation of test solution

[0221] 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.

[0222] (4) Detection and analysis

[0223] The sample solution and the standard working solution were tested by high performance liquid chromatography, and the corresponding chromatogram peak areas were measured. A standard curve was drawn with the concentration of the standard working solution as the abscissa and the chromatogram peak area as the ordinate. Based on the test results of the sample solution in step (3) (as shown in FIG3 , where 1 is L-methionine and 2 is citric acid), the concentrations of L-methionine and citric acid in the secretin extraction step were calculated in combination with the standard curve.

[0224] 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.

[0225] Table 1 Gradient elution program

[0226] 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.

[0227] (5) Calculate the number of colonies before inactivation of inactivated bacteria

[0228] Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

[0229] 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.

[0230] 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.

[0231] Comparative Example 1

[0232] This embodiment provides a fermentation-inactivated bacterium of Bifidobacterium lactis BL-99, the preparation method of which comprises the following steps:

[0233] 1. Preparation of tertiary seeds is the same as in Example 1.

[0234] 2. Preparation of fermented seeds, same as in Example 1.

[0235] 3. Fermentation broth culture, same as Example 1.

[0236] 4. Bacteria inactivation

[0237] Sterile purified water was used to mix the bacterial mud and resuspend it to obtain a mixed solution (concentration 3×10 11 cfu / mL), and the mixture was heat sterilized at temperatures of 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min.

[0238] Test Example 1: 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging

[0239] 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of mixed solution (10 9 cfu / 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:

[0240] Test Example 2: Hydroxyl Radical Scavenging

[0241] A total of 1.0 mL of the mixture (10 9cfu / 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:

[0242] In the present disclosure, 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 it can stably exist under different heat sterilization conditions within the experimental range (content change range ≤ 20%). It was found that 6 substances can be used as potential detection targets, namely: Proly-Alanine, L-Methionine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic peptide, GRPPK. At the same time, the 6 detection targets in the bacteriocin were detected, and it was found that only citric acid and L-methionine were present in the bacteriocin. The other four detection targets were not extracted or the content was below the detection line. Therefore, in the present disclosure, citric acid and L-methionine are used as detection targets to characterize the bacteriocin.

[0243] The test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG4 . As can be seen from FIG4 , when the extraction concentration is 3×10 11 cfu / mL, the extraction temperature was 4°C, and the extraction time was 1h, 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 shown in FIG5 , in which the extraction temperatures and times of K1-K9 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively; The extraction temperatures and times for K10-K18 were 4°C, 1 h, 4°C, 2 h, 4°C, 3 h, 25°C, 1 h, 25°C, 2 h, 25°C, 3 h, 37°C, 1 h, 37°C, 2 h, and 37°C, 3 h, respectively; and the extraction temperatures and times for K19-K27 were 4°C, 1 h, 4°C, 2 h, 4°C, 3 h, 25°C, 1 h, 25°C, 2 h, 25°C, 3 h, 37°C, 1 h, 37°C, 2 h, and 37°C, 3 h, respectively. As shown in Figure 5, the effects of the extraction conditions on DPPH radicals were relatively small.

[0244] The hydroxyl radical scavenging ability and DPPH radical scavenging ability of the postbiotics obtained in Comparative Example 1 are shown in FIG6 . The DPPH radical scavenging ability of the bacteriocin does not change much with temperature at first, but decreases significantly under high temperature conditions of 121° C.

[0245] The above description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations of the present disclosure are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure. Industrial Applicability

[0246] The bacteriocin of Bifidobacterium lactis BL-99, the fermented inactivated bacteria of Bifidobacterium lactis BL-99, and the postbiotics of Bifidobacterium lactis BL-99 disclosed in the present invention have antioxidant functions and can be applied to food and medicine. In addition, compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin of the present invention is a liquid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

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.

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

1.

4. A method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 1 to 3, 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.

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

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

7. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 6, characterized in that: The temperature of heat sterilization is 70-121°C; optionally, the temperature of heat sterilization is 70-100°C.

8. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 7, characterized in that: The heat sterilization time is 5-30 minutes; optionally, the heat sterilization time is 10-16 minutes.

9. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 8, characterized in that: The method further comprises a fermentation step: culturing Bifidobacterium lactis BL-99 in liquid, and stopping the fermentation when the Bifidobacterium lactis BL-99 grows to a logarithmic phase.

10. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 9, characterized in that: Optionally, the fermentation step includes: 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.

11. The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 10, characterized in that: Optionally, the preparation of the Bifidobacterium lactis BL-99 fermentation seed liquid comprises: activating the Bifidobacterium lactis BL-99 strain, purifying, culturing in a first-level seed liquid, multiplying in a second-level seed liquid, culturing in a third-level seed liquid, and preparing fermentation seeds to obtain the Bifidobacterium lactis BL-99 fermentation seed liquid; Optionally, the culture medium is MRS liquid culture medium; Optionally, the solvent is water; Optionally, the solvent is sterile water; Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

12. A method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99, characterized in that: The bacterial mud separated from the fermentation liquid is resuspended with a solvent to obtain a mixed liquid, and the mixed liquid is heat sterilized to obtain the Bifidobacterium lactis BL-99 fermentation-inactivated bacteria.

13. The method for preparing the fermented inactivated bacteria of Bifidobacterium lactis BL-99 according to claim 12, characterized in that: The heat sterilization temperature is 70-121° C., and the heat sterilization time is 5-30 minutes.

14. The method for preparing the fermented inactivated bacteria of Bifidobacterium lactis BL-99 according to claim 12 or 13, characterized in that: The total colony count of Bifidobacterium lactis BL-99 in the mixed solution was 0.5×10 11 -5×10 11 cfu / mL.

15. A fermented inactivated bacterium of Bifidobacterium lactis BL-99, characterized in that: Prepared by the method according to any one of claims 12 to 14.

16. A Bifidobacterium lactis BL-99 postbiotic, characterized in that The method comprises at least one of the secretory agent of Bifidobacterium lactis BL-99 according to any one of claims 1 to 3 and the fermented inactivated bacteria of Bifidobacterium lactis BL-99 according to claim 15.

17. Use of the Bifidobacterium lactis BL-99 postbiotic according to claim 16 in preparing food, characterized in that: The food comprises at least one of dairy products and beverages.

18. Use of the Bifidobacterium lactis BL-99 postbiotic according to claim 16 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.

19. Use of the Bifidobacterium lactis BL-99 postbiotic according to claim 16 in anti-oxidation.

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