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WO2025167388A1 - Bifidobacterium lactis bl-99 bacteriocin, inactivated bacteria, preparation method, and use thereof - Google Patents

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

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Publication number
WO2025167388A1
WO2025167388A1 PCT/CN2024/144022 CN2024144022W WO2025167388A1 WO 2025167388 A1 WO2025167388 A1 WO 2025167388A1 CN 2024144022 W CN2024144022 W CN 2024144022W WO 2025167388 A1 WO2025167388 A1 WO 2025167388A1
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WO
WIPO (PCT)
Prior art keywords
bifidobacterium lactis
fermentation
bacteriocin
preparing
liquid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/144022
Other languages
French (fr)
Chinese (zh)
Inventor
洪维鍊
蓝航莲
何剑
赵雯
曾照中
方冰
王然
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inner Mongolia Yili Industrial Group Co Ltd
Original Assignee
Inner Mongolia Yili Industrial Group Co Ltd
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Filing date
Publication date
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Publication of WO2025167388A1 publication Critical patent/WO2025167388A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • 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.
  • postbiotics are attracting much attention as a hot research field.
  • ISAPP International Scientific Association of Probiotics and Prebiotics
  • Postbiotics refer to preparations of inanimate microorganisms and/or their components that are beneficial to host health.
  • 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.
  • 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.
  • postbiotics are considered to be a potential functional food and health management method, and have received special attention from the industry.
  • 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.
  • 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.
  • 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.
  • 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 preservation number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.
  • the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:
  • Bacteriocin extraction using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG1 is a flow chart of the preparation of secretory agents from Bifidobacterium lactis BL-99 in Example 1;
  • FIG3 is a liquid chromatogram of a bacteriocin sample obtained in Example 1;
  • Figure 5 shows the DPPH radical scavenging ability of secretory agents of Bifidobacterium lactis BL-99 under different extraction conditions
  • 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.
  • 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 preservation number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.
  • 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.
  • 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.
  • 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.
  • the mass ratio of citric acid to L-methionine in the bacteriocin is 8-9:1.
  • 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.
  • 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;
  • 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.
  • 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.
  • the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:
  • Bacteriocin extraction using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;
  • 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.
  • 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.
  • 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 any value between 0.5 ⁇ 10 11 and 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.
  • 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.
  • 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.
  • 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;
  • 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;
  • the preparation of the Bifidobacterium lactis BL-99 fermentation seed liquid includes: 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;
  • the culture medium is MRS liquid culture medium.
  • the solvent is water; optionally, the solvent is sterile water, specifically sterile purified water;
  • the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • the secretin of Bifidobacterium lactis BL-99 is prepared by the following steps:
  • the cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.
  • the third-level seeds After the third-level seeds are prepared, they can be placed at 4°C for no more than 6 hours.
  • Indicators for determining the end point of seed growth at each level pH 4.2-4.6, OD600 ⁇ 2.0.
  • 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.
  • the fermentation broth can be cultured or cooled to 10-20°C and stored for no more than 6 hours.
  • the fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.
  • centrifugation can be performed or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.
  • the collected bacterial sludge was transferred to a sterile fermentation tank for extraction.
  • the bacterial sludge is extracted and sterile purified water is mixed with the bacterial sludge to obtain a mixed solution, wherein the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 0.5 ⁇ 10 11 -5 ⁇ 10 11 cfu/mL, the extraction temperature is 0-37° C., and the extraction time is 0.5-3 h.
  • 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.
  • the filled bottled supernatant was heat sterilized to obtain the bacteriocin.
  • the sterilization conditions for each strain were as follows:
  • the sterilization conditions of Bifidobacterium lactis BL-99 are 70-121°C, 5-30 min.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 any value between 0.5 ⁇ 10 11 and 5 ⁇ 10 11 cfu/mL.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.
  • the third-level seeds After the third-level seeds are prepared, they can be placed at 4°C for no more than 6 hours.
  • Indicators for determining the end point of seed growth at each level pH 4.2-4.6, OD600 ⁇ 2.0.
  • 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.
  • the fermentation broth can be cultured or cooled to 10-20°C and stored for no more than 6 hours.
  • Seed tank fermentation endpoint determination indicators pH 4.2-4.6, OD600 ⁇ 2.0.
  • pH value 6.2-6.4, sterilization conditions: 121°C, 15-20min.
  • the fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.
  • centrifugation can be performed or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.
  • pH value 6.2-6.4, sterilization conditions: 121°C, 15-20min.
  • the collected bacterial sludge was transferred to a sterile fermentation tank for extraction.
  • the bacterial sludge was extracted, and sterile purified water was mixed with the bacterial sludge to obtain a mixed solution, wherein the total colony counts of Bifidobacterium lactis BL-99 in the mixed solution were 1 ⁇ 10 11 cfu/mL, 1.5 ⁇ 10 11 cfu/mL, and 3 ⁇ 10 11 cfu/mL, respectively; the extraction temperatures were 4° C., 25° C., and 37° C., respectively; the extraction times were 1 h, 2 h, and 3 h, respectively; and the extraction speed was 70 rpm.
  • 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 .
  • 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:
  • 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 ⁇ g/mL, and store at 4°C.
  • 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.
  • 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.
  • 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.
  • 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.
  • the concentrations of L-methionine and citric acid in the secretin extraction step were calculated in combination with the standard curve.
  • Chromatographic analysis conditions An LC-20A analysis system was used, with a Poroshell 120Aq-C18 column (4.6 mm ⁇ 150 mm, 2.7 ⁇ m); mobile phase A was 0.1% phosphoric acid in water; mobile phase B was 0.1% phosphoric acid in acetonitrile; the gradient elution program was 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 ⁇ L.
  • This embodiment provides a fermentation-inactivated bacterium of Bifidobacterium lactis BL-99, the preparation method of which comprises the following steps:
  • Sterile purified water was used to mix the bacterial sludge and resuspend the mixture to obtain a mixed solution (concentration 3 ⁇ 10 11 cfu/mL), and the mixed solution was heat sterilized at temperatures of 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min.
  • Test Example 1 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging
  • LC-MS was used to detect the components of the inactivated bacteria obtained in Comparative Example 1, and the components with peak areas greater than 10 4 in the test results were screened to obtain targets for characterizing the components of the inactivated bacteria.
  • the test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG4 .
  • the extraction concentration is 3 ⁇ 10 11 cfu/mL
  • the extraction temperature is 4°C
  • the extraction time is 1 h
  • the hydroxyl radical scavenging ability of the bacteriocin is better
  • the test results of the DPPH radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG5 .
  • the extraction temperature and time of K1-K9 are 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;
  • 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
  • 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.
  • 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.
  • 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.

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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

乳双歧杆菌BL-99菌泌素、灭活菌、制备方法及其应用Bifidobacterium lactis BL-99 secretory factor, inactivated bacteria, preparation method and application thereof

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本公开要求于2024年02月07日提交中国专利局的申请号为202410174812.1、名称为“乳双歧杆菌BL-99菌泌素、制备方法及其抗氧化应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。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.

技术领域Technical Field

本公开涉及微生物技术领域，具体而言，涉及乳双歧杆菌BL-99菌泌素、灭活菌、制备方法及其应用。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.

背景技术Background Art

当下，后生元作为一个热门研究领域备受关注。2021年国际益生菌和益生元科学协会(ISAPP)发表了后生元的共识声明，后生元(postbiotics)是指对宿主健康有益的无生命微生物和/或其成分的制剂。作为益生元和益生菌的衍生物，后生元具有多重潜在益处。它们能够调节肠道菌群、增强肠道屏障功能、调节肠道炎症反应等，继而对人体肠道健康产生积极影响。此外，后生元的生物活性不仅局限于肠道，它还具有抑制口腔致病菌、调节肺部炎症反应等生物活性。随着人们对肠道健康和微生物组的认识不断深入，后生元被认为是一种潜在的功能性食品和健康管理手段，受到了产业界的格外关注。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.

目前，针对后生元生物活性的报道主要参考益生菌或菌体的生物活性，乳双歧杆菌BL-99属于较为典型的后生元菌株，前期研究发现乳双歧杆菌BL-99在调节肠道菌群、调节血压和调节睡眠方面具有潜力，如CN201811161053.6公开乳双歧杆菌BL-99具有调节胃肠道菌群功效。但其产品具体形态和制备方式均会对其性能有所影响。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.

鉴于此，特提出本公开。In view of this, the present disclosure is proposed.

发明内容Summary of the Invention

本公开的目的在于提供乳双歧杆菌BL-99菌泌素、灭活菌、制备方法及其应用，得到具有抗氧化性能的产品。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.

本公开所述的乳双歧杆菌BL-99菌泌素是指对乳双歧杆菌BL-99中分离出的菌泥中乳双歧杆菌BL-99的水溶性代谢产物和乳双歧杆菌BL-99的胞吐物进行提取得到的产品。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.

本公开是这样实现的：The present disclosure is achieved as follows:

第一方面，本公开提供一种乳双歧杆菌BL-99菌泌素，包括乳双歧杆菌BL-99的胞吐物及乳双歧杆菌BL-99的代谢产物，所述菌泌素中柠檬酸与L-蛋氨酸质量比大于2：1，所述乳双歧杆菌BL-99的保藏编号CGMCC No.15650。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 preservation number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.

第二方面，本公开提供一种前述乳双歧杆菌BL-99菌泌素，包括以下步骤：In a second aspect, the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:

菌泌素提取，采用溶剂对发酵液中分离出的菌泥进行提取，得到混合液；Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

灭菌，分离出所述混合液中的上清液并进行热灭菌，得到所述菌泌素。Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

第三方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在制备食品中的应用，所述食品包括乳制品、饮料中的至少一种。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.

第四方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在制备组合物中的应用，所述组合物包括抗氧化组合物；优选的，所述组合物选自药物、保健食品和饲料中得至少一种。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.

第五方面，本公开提供一种乳双歧杆菌BL-99发酵灭活菌的制备方法，采用溶剂对发酵液中分离出的菌泥进行重悬得到混合液，对所述混合液进行热灭菌，得到所述乳双歧杆菌BL-99发酵灭活菌。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.

第六方面，本公开提供一种乳双歧杆菌BL-99发酵灭活菌，由前述实施方式任意一项所述方法制备得到。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.

第七方面，本公开提供一种乳双歧杆菌BL-99后生元，包括前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素和前述实施方式所述的乳双歧杆菌BL-99发酵灭活菌中的至少一种。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.

第八方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99后生元在制备食品中的应用，所述食品包括乳制品、饮料中的至少一种。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.

第九方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99后生元在制备组合物中的应用，所述组合物包括抗氧化组合物；优选的，所述组合物选自药物、保健食品和饲料中得至少一种。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.

第十方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在抗氧化中的应用。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.

本公开具有以下有益效果：The present disclosure has the following beneficial effects:

本公开中的乳双歧杆菌BL-99菌泌素具有抗氧化的功能，可应用于食品和药品中，且相比于包括完整死细胞+细胞壁组分+细胞膜组分+无细胞上清的混合体，本公开中的菌泌素为液体或水溶性粉末，在一些液体饮料、药品中使用更有优势。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.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。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.

图1为实施例1中乳双歧杆菌BL-99菌泌素制备的流程图；FIG1 is a flow chart of the preparation of secretory agents from Bifidobacterium lactis BL-99 in Example 1;

图2为实施例1中乳双歧杆菌BL-99菌泌素的实物图；FIG2 is a physical image of the secretory activity of Bifidobacterium lactis BL-99 in Example 1;

图3实施例1得到菌泌素样品的液相色谱图；FIG3 is a liquid chromatogram of a bacteriocin sample obtained in Example 1;

图4为不同提取条件下乳双歧杆菌BL-99菌泌素的羟自由基清除能力；FIG4 shows the hydroxyl radical scavenging ability of secretin from Bifidobacterium lactis BL-99 under different extraction conditions;

图5为不同提取条件下乳双歧杆菌BL-99菌泌素的DPPH自由基清除能力；Figure 5 shows the DPPH radical scavenging ability of secretory agents of Bifidobacterium lactis BL-99 under different extraction conditions;

图6为对比例1中灭活菌在不同热灭菌温度下的羟自由基清除能力和DPPH自由基清除能力。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.

具体实施方式DETAILED DESCRIPTION

为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。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.

第一方面，本公开提供一种乳双歧杆菌BL-99菌泌素，包括乳双歧杆菌BL-99的胞吐物及乳双歧杆菌BL-99的代谢产物，所述菌泌素中柠檬酸与L-蛋氨酸质量比大于2：1，所述乳双歧杆菌BL-99的保藏编号CGMCC No.15650。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 preservation number of Bifidobacterium lactis BL-99 is CGMCC No. 15650.

本公开中，菌泌素中主要包括胞吐物和菌体代谢产物等组分，随着对菌泌素提取的进行，菌泌素中的柠檬酸和L-蛋氨酸的浓度逐渐增加，因此，本公开中以L-蛋氨酸和柠檬酸的浓度表征菌泌素。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.

本公开中的乳双歧杆菌BL-99菌泌素具有抗氧化的功能，可应用于食品和药品中，且相比于包括完整死细胞+细胞壁组分+细胞膜组分+无细胞上清的混合体，本公开中的菌泌素为液体或水溶性粉末，在一些液体饮料、药品中使用更有优势。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.

在可选的实施方式中，所述菌泌素中柠檬酸与L-蛋氨酸质量比为2-25：1，具体地，可以为2：1、5：1、10：1、15：1、20：1、25：1或2-25：1之间的任意值，或＞25：1的任意值。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.

可选地，所述菌泌素中柠檬酸与L-蛋氨酸质量比为8-9：1。Optionally, the mass ratio of citric acid to L-methionine in the bacteriocin is 8-9:1.

在可选的实施方式中，所述菌泌素中L-蛋氨酸浓度为0.2-1mg/ml、柠檬酸浓度为2-5mg/ml。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.

具体地，本公开中菌泥和上清液的分离可以采用现有的分离方式，例如选择离心的方式实现固液分离；一些实施例中，菌泌素中L-蛋氨酸浓度为0.2mg/ml、0.3mg/ml、0.4mg/ml、0.51mg/ml、0.6mg/ml、0.7mg/ml、0.8mg/ml、0.9mg/ml、1mg/ml或0.2-1mg/ml之间的任意值，或＞1mg/ml的任意值；柠檬酸浓度为2mg/ml、3mg/ml、5mg/ml、4mg/ml、4.5mg/ml、5mg/ml或2-5mg/ml之间的任意值，或＞5mg/ml的任意值。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.

可选地，所述菌泌素中L-蛋氨酸浓度为0.25-0.3mg/ml、柠檬酸浓度为2-2.8mg/ml。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.

第二方面，本公开提供一种前述乳双歧杆菌BL-99菌泌素，包括以下步骤：In a second aspect, the present disclosure provides the aforementioned Bifidobacterium lactis BL-99 secretin, comprising the following steps:

菌泌素提取，采用溶剂对发酵液中分离出的菌泥进行提取，得到混合液；Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

灭菌，分离出所述混合液中的上清液并进行热灭菌，得到所述菌泌素。Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

在可选的实施方式中，提取温度为0-37℃，具体地可以为0℃、2℃、4℃、6℃、8℃、10℃、15℃、20℃、25℃、30℃、37℃或0-37℃之间的任意值；可选地，提取温度为3-5℃。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.

在可选的实施方式中，提取时间0.5-3h，具体地可以为0.5h、1h、1.5h、2h、2.5h、3h或1-3h之间的任意值；可选地，提取时间30-90min。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.

在可选的实施方式中，所述混合液中乳双歧杆菌BL-99的菌落总数为0.5×1011-5×1011，具体地可以为0.5×1011cfu/mL、0.7×1011cfu/mL、0.9×1011cfu/mL、1×1011cfu/mL、2×1011cfu/mL、3×1011cfu/mL、4×1011cfu/mL、5×1011cfu/mL或0.5×1011-5×1011cfu/mL之间的任意值；可选地，所述混合液中乳双歧杆菌BL-99的菌落总数为2×1011-4×1011cfu/mL。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 any value between 0.5×10 11 and 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.

在可选的实施方式中，热灭菌的温度为70-121℃，具体地可以为70℃、80℃、90℃、100℃、110℃、121℃或70-121℃之间的任意值；可选地，热灭菌的温度为70-100℃。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.

在可选的实施方式中，热灭菌的时间为5-30min，具体地可以为5min、10min、15min、20min、25min、30min或5-30min之间的任意值；可选地，热灭菌的时间为10-16min。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.

在可选的实施方式中，还包括发酵步骤：将乳双歧杆菌BL-99进行液体培养，待生长到对数期时，停止发酵；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;

可选地，所述发酵步骤包括：将乳双歧杆菌BL-99发酵种子液接种至培养基中，先在温度30-40℃、转速60-80rpm、pH 5.8-6.2条件下发酵14-16h，得到乳双歧杆菌BL-99发酵液；可选地，所述乳双歧杆菌BL-99发酵种子液的制备包括：将乳双歧杆菌BL-99菌株经活化、纯化、一级种子液培养、二级种子液扩繁、三级种子液培养和发酵种子制备，得到所述乳双歧杆菌BL-99发酵种子液；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; Optionally, the preparation of the Bifidobacterium lactis BL-99 fermentation seed liquid includes: 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;

可选地，所述培养基为MRS液体培养基。Optionally, the culture medium is MRS liquid culture medium.

在可选的实施方式中，所述溶剂为水；可选地，所述溶剂为无菌水，具体地可以选择无菌纯化水；In an optional embodiment, the solvent is water; optionally, the solvent is sterile water, specifically sterile purified water;

可选地，所述灭菌步骤后，还对菌泌素进行冻干，得到冻干菌泌素。Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

在可选的实施方式中，所述菌泌素中L-蛋氨酸浓度为0.2-1mg/ml、柠檬酸浓度为2-5mg/ml。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.

具体地，本公开中菌泥和上清液的分离可以采用现有的分离方式，例如选择离心的方式实现固液分离；一些实施例中，菌泌素中L-蛋氨酸浓度为0.2mg/ml、0.3mg/ml、0.4mg/ml、0.51mg/ml、0.6mg/ml、0.7mg/ml、0.8mg/ml、0.9mg/ml、1mg/ml或0.2-1mg/ml之间的任意值，或＞1mg/ml的任意值；柠檬酸浓度为2mg/ml、3mg/ml、5mg/ml、4mg/ml、4.5mg/ml、5mg/ml或2-5mg/ml之间的任意值，或＞5mg/ml的任意值。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.

可选地，所述菌泌素中L-蛋氨酸浓度为0.25-0.3mg/ml、柠檬酸浓度为2-2.8mg/ml。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.

当灭菌步骤后对菌泌素进行冻干，得到冻干菌泌素，其中的溶剂被去除，所述冻干菌泌素中柠檬酸与L-蛋氨酸质量比大于2：1。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.

具体地，一些实施例中，乳双歧杆菌BL-99菌泌素由以下步骤制备得到：Specifically, in some embodiments, the secretin of Bifidobacterium lactis BL-99 is prepared by the following steps:

1.三级种子制备1. Preparation of Tertiary Seeds

1.1标准冻存管1.1 Standard cryopreservation tubes

由纯化后的菌种统一制备，分装成1.5mL离心管，不少于50支，-80℃冰箱存放，保存期限不超过6个月。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.

1.2冻存管活化1.2 Activation of cryotubes

取-80℃保藏菌种一份，室温下解冻，无菌取200μL菌液接入10mL种子液体培养基，37℃静置培养11-13h。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.

1.3一次纯化1.3 Primary purification

取培养好的菌液进行稀释涂布，稀释度为-4、-5、-6，每个稀释度制作2个MRS固体平板，37℃倒置培养48h-72h至平板上形成明显菌落，用接种环分别挑取单菌落于5管10mLMRS液体培养基中，挑取菌落大小均一，37℃静置培养11-13h。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.

1.4二次纯化1.4 Secondary purification

取培养好的一次纯化菌液进行稀释涂布，稀释度为-4、-5、-6，每个稀释度制作2个MRS固体平板，37℃倒置培养48h-72h至平板上形成明显菌落，用接种环分别挑取单菌落于5管10mLMRS液体培养基中，挑取菌落大小均一，37℃静置培养11-13h。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.

1.5一级种子制备1.5 Primary seed preparation

选择1管培养好的二次纯化菌液，用移液枪分别吸取200μL菌液至5管10mL MRS液体培养基内，37℃静置培养11-13h。Select one tube of secondary purified bacterial culture, and use a pipette to 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.

1.6二级种子制备1.6 Secondary seed preparation

选择4管培养好一级种子，分别吸取4mL注入4瓶80mLMRS液体培养基内，37℃静置培养11-13h。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.

1.7三级种子制备1.7 Preparation of tertiary seeds

培养好的二级种子，分别倒入2瓶1.8LMRS液体培养基内，37℃静置培养12-14h。The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

1.8三级种子暂存1.8 Temporary storage of third-level seeds

三级种子制备完成可4℃放置不超过6h。After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

1.9过程质量控制1.9 Process Quality Control

(1)各级种子生长终点判定指标：pH 4.2-4.6，OD600≥2.0。(1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

(2)纯度检测：显微镜观察菌体形态，100倍油镜下菌体形态完整，呈杆菌，不成链，略有弯曲，呈弧形。(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.

(3)污染物检测：检测三级种子液，包含大肠杆菌、非乳酸菌。(3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

2.发酵种子制备2. Fermentation Seed Preparation

2.1接种2.1 Vaccination

(1)打开搅拌桨和控温程序，转速70rpm，温度37℃。(1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

(2)打开氮气入口阀门，向发酵罐内通入小气流氮气，使发酵罐保持正压，持续5-10min。(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.

(3)接种时向接种环上倾入酒精并点燃，使之形成火焰圈。(3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

(4)旋开接种器上口，在火焰上方无菌区倾入培养好的种子，接种量2.5％。(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%.

(5)接种后旋紧接种器上盖，并关闭接种阀，熄灭酒精火焰环。(5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

(6)关闭氮气进气阀、排气阀，控制罐压为0.01-0.03MPa进行保压发酵。(6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

2.2发酵2.2 Fermentation

(1)设定发酵参数，转速70rpm，温度37℃。(1) Set the fermentation parameters to 70 rpm and 37°C.

(2)发酵时间11h-13h，每隔2h进行发酵状态监控，包括pH、OD600、温度、转速。(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.

(3)发酵结束即可进行发酵液培养，或降温至10-20℃保存不超过6h。(3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

2.3过程质量控制2.3 Process quality control

(1)种子罐发酵终点判定指标：pH 4.2-4.6，OD600≥2.0。(1) Indicators for determining the end point of seed tank fermentation: pH 4.2-4.6, OD600 ≥ 2.0.

(2)纯度检测：显微镜观察种子罐终点发酵液菌体形态，100倍油镜下菌体形态完整，呈杆菌，不成链，略有弯曲，呈弧形。(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.

(3)污染物检测：检测种子罐终点发酵液，包含大肠杆菌、非乳酸菌。(3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

3.发酵液培养3. Fermentation Broth Culture

3.1接种3.1 Vaccination

(1)打开搅拌桨和控温程序，转速70rpm，温度37℃。(1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

(2)对接种管道进行蒸汽灭菌，持续30min。(2) Steam sterilize the inoculation pipe for 30 minutes.

(3)打开氮气进气阀，充氮气5-10min，打开种子罐底阀和发酵罐接种阀进行接种，接种量2％。(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%.

(4)接种完毕后关闭发酵罐接种阀，对接种管道进行清洗。(4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

(5)关闭氮气进气阀、排期阀，控制罐压为0.01-0.03MPa进行保压发酵。(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.

3.2发酵3.2 Fermentation

(1)设定发酵参数，温度30-40℃、转速60-80rpm，恒pH值5.8-6.2发酵。(1) Set the fermentation parameters to 30-40°C, 60-80 rpm, and a constant pH of 5.8-6.2.

(2)发酵时间14-16h，每隔2h进行发酵状态监控，包括pH、OD600、温度、转速。(2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

(3)发酵结束后即可进行离心操作，或降温至10-20℃保存不超过4h。(3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

3.3菌泥分离3.3 Mushroom sludge separation

(1)开启离心机操作水供应阀，操作水管道总压>3bar，机封水压力保持1.8-2.5bar。(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.

(2)启动离心机，等待离心机转速达到11600-11800rpm时，程序自动进行排渣操作，同时主界面转鼓指示呈常亮状态。(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.

(3)打开进料阀，乳双歧杆菌BL-99发酵液进入离心机转鼓开始离心，得到菌泥。(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.

(4)离心过程参数设定：进料速度600L/h，排渣时间200s。(4) Centrifugal process parameter settings: feed rate 600 L/h, slag discharge time 200 s.

4.菌泌素提取4. Bacteriocin Extraction

4.1菌泥转移4.1 Mud transfer

将收集菌泥转移至无菌发酵罐中，进行提取。The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

4.2提取条件4.2 Extraction conditions

对所述菌泥进行提取，采用无菌纯化水与所述菌泥混合得到混合液，混合液中乳双歧杆菌BL-99的菌落总数为0.5×1011-5×1011cfu/mL、提取温度0-37℃、提取时间0.5-3h。The bacterial sludge is extracted and sterile purified water is mixed with the bacterial sludge to obtain a mixed solution, wherein the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 0.5×10 11 -5×10 11 cfu/mL, the extraction temperature is 0-37° C., and the extraction time is 0.5-3 h.

需要说明的是，在实际生产过程中，可以调整3.2步骤中副干酪乳杆菌K56发酵液的OD600的值，同时控制提取步骤中无菌纯化水的加入量，使得混合液的体积为副干酪乳杆菌K56发酵液体积的1％，以更方便的调整混合液中副干酪乳杆菌K56的菌落总数。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.

5.离心分离5. Centrifugal separation

(1)开启离心机操作水供应阀，操作水管道总压>3bar，机封水压力保持1.8-2.5bar。(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.

(2)启动离心机，等待离心机转速达到11600-11800rpm时，程序自动进行排渣操作，同时主界面转鼓指示呈常亮状态。(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.

(3)打开进料阀，混合液进入离心机转鼓开始离心。(3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

(4)离心过程参数设定：进料速度600L/h，排渣时间200s。(4) Centrifugal process parameter settings: feed rate 600 L/h, slag discharge time 200 s.

(5)将离心上清液转移至收集罐中。(5) Transfer the centrifuged supernatant to a collection tank.

6.分装灭菌6. Packaging and sterilization

将灌装好的瓶装上清液进行热灭菌得到菌泌素，各个菌株灭菌条件如下：The filled bottled supernatant was heat sterilized to obtain the bacteriocin. The sterilization conditions for each strain were as follows:

乳双歧杆菌BL-99灭菌条件为70-121℃，5-30min。The sterilization conditions of Bifidobacterium lactis BL-99 are 70-121°C, 5-30 min.

第三方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在制备食品中的应用，所述食品包括乳制品、饮料中的至少一种。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.

第四方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在制备组合物中的应用，所述组合物包括抗氧化组合物；优选的，所述组合物选自药物、保健食品和饲料中得至少一种。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.

第五方面，本公开提供一种乳双歧杆菌BL-99发酵灭活菌的制备方法，采用溶剂对发酵液中分离出的菌泥进行重悬得到混合液，对所述混合液进行热灭菌，得到所述乳双歧杆菌BL-99发酵灭活菌。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.

在可选实施方式中，所述热灭菌的温度为70-121℃，具体地可以为70℃、80℃、90℃、100℃、110℃、121℃或70-121℃之间的任意值；热灭菌时间为5-30min，具体地可以为5min、10min、15min、20min、25min、30min或5-30min之间的任意值。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.

在可选实施方式中，所述混合液中乳双歧杆菌BL-99的菌落总数为0.5×1011-5×1011cfu/mL，具体地可以为0.5×1011cfu/mL、0.7×1011cfu/mL、0.9×1011cfu/mL、1×1011cfu/mL、2×1011cfu/mL、3×1011cfu/mL、4×1011cfu/mL、5×1011cfu/mL或0.5×1011-5×1011cfu/mL之间的任意值。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 any value between 0.5×10 11 and 5×10 11 cfu/mL.

第六方面，本公开提供一种乳双歧杆菌BL-99发酵灭活菌，由前述实施方式任意一项所述方法制备得到。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.

第七方面，本公开提供一种乳双歧杆菌BL-99后生元，包括前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素和前述实施方式所述的乳双歧杆菌BL-99发酵灭活菌中的至少一种。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.

第八方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99后生元在制备食品中的应用，所述食品包括乳制品、饮料中的至少一种。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.

第九方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99后生元在制备组合物中的应用，所述组合物包括抗氧化组合物；优选的，所述组合物选自药物、保健食品和饲料中得至少一种。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.

第十方面，本公开提供一种前述实施方式任意一项所述的乳双歧杆菌BL-99菌泌素在抗氧化中的应用。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.

以下结合实施例对本公开的特征和性能作进一步的详细描述。The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

实施例1Example 1

本实施例提供一种乳双歧杆菌BL-99菌泌素，如图1所示，其制备方法包括以下步骤：This embodiment provides a secretin of Bifidobacterium lactis BL-99, as shown in FIG1 , and its preparation method comprises the following steps:

1.三级种子制备1. Preparation of Tertiary Seeds

1.1标准冻存管1.1 Standard cryopreservation tubes

由纯化后的菌种统一制备，分装成1.5mL离心管，不少于50支，-80℃冰箱存放，保存期限不超过6个月。Prepare uniformly from the purified strains, package into 1.5mL centrifuge tubes, no less than 50, and store in a -80℃ refrigerator with a shelf life of no more than 6 months.

1.2冻存管活化1.2 Activation of cryotubes

取-80℃保藏菌种一份，室温下解冻，无菌取200μL菌液接入10mL种子液体培养基，37℃静置培养11-13h。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.

1.3一次纯化1.3 Primary purification

取培养好的菌液进行稀释涂布，稀释度为-4、-5、-6，每个稀释度制作2个MRS固体平板，37℃倒置培养48h-72h至平板上形成明显菌落，用接种环分别挑取单菌落于5管10mLMRS液体培养基中，挑取菌落大小均一，37℃静置培养11-13h。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.

1.4二次纯化1.4 Secondary purification

取培养好的一次纯化菌液进行稀释涂布，稀释度为-4、-5、-6，每个稀释度制作2个MRS固体平板，37℃倒置培养48h-72h至平板上形成明显菌落，用接种环分别挑取单菌落于5管10mLMRS液体培养基中，挑取菌落大小均一，37℃静置培养11-13h。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.

1.5一级种子制备1.5 Primary seed preparation

选择1管培养好的二次纯化菌液，用移液枪分别吸取200μL菌液至5管10mL MRS液体培养基内，37℃静置培养11-13h。Select one tube of secondary purified bacterial culture, and use a pipette to 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.

1.6二级种子制备1.6 Secondary seed preparation

选择4管培养好一级种子，分别吸取4mL注入4瓶80mLMRS液体培养基内，37℃静置培养11-13h。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.

1.7三级种子制备1.7 Preparation of tertiary seeds

培养好的二级种子，分别倒入2瓶1.8LMRS液体培养基内，37℃静置培养12-14h。The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

1.8三级种子暂存1.8 Temporary storage of third-level seeds

三级种子制备完成可4℃放置不超过6h。After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

1.9过程质量控制1.9 Process Quality Control

(1)各级种子生长终点判定指标：pH 4.2-4.6，OD600≥2.0。(1) Indicators for determining the end point of seed growth at each level: pH 4.2-4.6, OD600 ≥ 2.0.

(2)纯度检测：显微镜观察菌体形态，100倍油镜下菌体形态完整，呈杆菌，不成链，略有弯曲，呈弧形。(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.

(3)污染物检测：检测三级种子液，包含大肠杆菌、非乳酸菌。(3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

三级种子发酵培养基配方
Formula of three-stage seed fermentation medium

pH值：6.2-6.4，灭菌条件：121℃，15-20minpH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min

2.发酵种子制备2. Fermentation Seed Preparation

2.1接种2.1 Vaccination

(1)打开搅拌桨和控温程序，转速70rpm，温度37℃。(1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

(2)打开氮气入口阀门，向发酵罐内通入小气流氮气，使发酵罐保持正压，持续5-10min。(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.

(3)接种时向接种环上倾入酒精并点燃，使之形成火焰圈。(3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

(4)旋开接种器上口，在火焰上方无菌区倾入培养好的种子，接种量2.5％。(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%.

(5)接种后旋紧接种器上盖，并关闭接种阀，熄灭酒精火焰环。(5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

(6)关闭氮气进气阀、排气阀，控制罐压为0.01-0.03MPa进行保压发酵。(6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

2.2发酵2.2 Fermentation

(1)设定发酵参数，转速70rpm，温度37℃。(1) Set the fermentation parameters to 70 rpm and 37°C.

(2)发酵时间11h-13h，每隔2h进行发酵状态监控，包括pH、OD600、温度、转速。(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.

(3)发酵结束即可进行发酵液培养，或降温至10-20℃保存不超过6h。(3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

2.3过程质量控制2.3 Process quality control

(1)种子罐发酵终点判定指标：pH 4.2-4.6，OD600≥2.0。(1) Seed tank fermentation endpoint determination indicators: pH 4.2-4.6, OD600 ≥ 2.0.

(2)纯度检测：显微镜观察种子罐终点发酵液菌体形态，100倍油镜下菌体形态完整，呈杆菌，不成链，略有弯曲，呈弧形。(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.

(3)污染物检测：检测种子罐终点发酵液，包含大肠杆菌、非乳酸菌。(3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

种子罐发酵培养基配方
Seed tank fermentation medium formula

pH值：6.2-6.4，灭菌条件：121℃，15-20min。pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

3.发酵液培养3. Fermentation Broth Culture

3.1接种3.1 Vaccination

(1)打开搅拌桨和控温程序，转速70rpm，温度37℃。(1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

(2)对接种管道进行蒸汽灭菌，持续30min。(2) Steam sterilize the inoculation pipe for 30 minutes.

(3)打开氮气进气阀，充氮气5-10min，打开种子罐底阀和发酵罐接种阀进行接种，接种量2％。(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%.

(4)接种完毕后关闭发酵罐接种阀，对接种管道进行清洗。(4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

(5)关闭氮气进气阀、排期阀，控制罐压为0.01-0.03MPa进行保压发酵。(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.

3.2发酵3.2 Fermentation

(1)设定发酵参数，温度37℃、转速70rpm，恒pH值6发酵。(1) Set the fermentation parameters to 37°C, 70 rpm, and a constant pH of 6.

(2)发酵时间14-16h，每隔2h进行发酵状态监控，包括pH、OD600、温度、转速。(2) The fermentation time was 14-16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

(3)发酵结束后即可进行离心操作，或降温至10-20℃保存不超过4h。(3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

发酵罐发酵培养基配方
Fermentation medium formula for fermentation tank

pH值：6.2-6.4，灭菌条件：121℃，15-20min。pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

3.3菌泥分离3.3 Mushroom sludge separation

(1)开启离心机操作水供应阀，操作水管道总压>3bar，机封水压力保持1.8-2.5bar。(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.

(2)启动离心机，等待离心机转速达到11600-11800rpm时，程序自动进行排渣操作，同时主界面转鼓指示呈常亮状态。(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.

(3)打开进料阀，乳双歧杆菌BL-99发酵液进入离心机转鼓开始离心，得到菌泥。(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.

(4)离心过程参数设定：进料速度600L/h，排渣时间200s。(4) Centrifugal process parameter settings: feed rate 600 L/h, slag discharge time 200 s.

4.菌泌素提取4. Bacteriocin Extraction

4.1菌泥转移4.1 Mud transfer

将收集菌泥转移至无菌发酵罐中，进行提取。The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

4.2提取条件4.2 Extraction conditions

对所述菌泥进行提取，采用无菌纯化水与所述菌泥混合得到混合液，混合液中乳双歧杆菌BL-99的菌落总数分别为1×1011cfu/mL、1.5×1011cfu/mL、3×1011cfu/mL、提取温度分别为4℃、25℃、37℃、提取时间分别为1h、2h、3h，提取转速70rpm。The bacterial sludge was extracted, and sterile purified water was mixed with the bacterial sludge to obtain a mixed solution, wherein the total colony counts of Bifidobacterium lactis BL-99 in the mixed solution were 1×10 11 cfu/mL, 1.5×10 11 cfu/mL, and 3×10 11 cfu/mL, respectively; the extraction temperatures were 4° C., 25° C., and 37° C., respectively; the extraction times were 1 h, 2 h, and 3 h, respectively; and the extraction speed was 70 rpm.

5.离心分离5. Centrifugal separation

(1)开启离心机操作水供应阀，操作水管道总压>3bar，机封水压力保持1.8-2.5bar。(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.

(2)启动离心机，等待离心机转速达到11600-11800rpm时，程序自动进行排渣操作，同时主界面转鼓指示呈常亮状态。(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.

(3)打开进料阀，混合液进入离心机转鼓开始离心。(3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

(4)离心过程参数设定：进料速度600L/h，排渣时间200s。(4) Centrifugal process parameter settings: feed rate 600 L/h, slag discharge time 200 s.

(5)将离心上清液转移至收集罐中。(5) Transfer the centrifuged supernatant to a collection tank.

6.分装灭菌6. Packaging and sterilization

将灌装好的瓶装上清液进行热灭菌得到菌泌素，乳双歧杆菌BL-99灭菌条件为75℃，10min，得到的乳双歧杆菌BL-99菌泌素如图2所示。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 .

7.定量检测7. Quantitative Detection

对实施例1得到的产品中L-蛋氨酸和柠檬酸的浓度进行检测，检测方法包括以下步骤：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:

(1)制备标准工作溶液(1) Preparation of standard working solution

标准储备液：分别单独精确称取适量标准品(即L-蛋氨酸和柠檬酸，精确至0.1mg)，加水溶解并分别配制成浓度为5mg/mL的标准储备液，置于-20℃下保存。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.

混合标准中间液：分别准确吸取适量体积的标准储备液，用水定容，配制成浓度为500μg/mL的混合标准中间液，于4℃冷藏保存。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.

混合标准工作液：根据需要用水逐级稀释混合标准中间溶液，配制成浓度分别为1μg/mL、5μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL、200μg/mL的混合标准工作溶液，现用现配。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.

(2)制备洗脱溶液(2) Preparation of elution solution

0.1％磷酸水溶液：取磷酸1mL，用水稀释并定容至1000mL，混匀，现配现用。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.

0.1％磷酸乙腈溶液：取磷酸1mL，用乙腈稀释并定容至1000mL，混匀，现配现用。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.

(3)制备供试品溶液(3) Preparation of test solution

液体样品：将乳双歧杆菌BL-99菌泌素样品混匀后直接吸取1mL，于4℃下10000rpm离心10min，将上清液稀释至线性范围内，上柱分析。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.

(4)检测分析(4) Detection and analysis

采用高效液相色谱法测试样品溶液和标准工作溶液，测定相应的色谱图峰面积，以标准工作液的浓度为横坐标，以色谱图峰面积为纵坐标，绘制标准曲线。根据步骤(3)中样品溶液的检测结果(如图3，其中1为L-蛋氨酸、2为柠檬酸)，结合标准曲线计算菌泌素中提取步骤中L-蛋氨酸和柠檬酸的浓度。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.

色谱分析条件：使用LC-20A分析系统，色谱柱为Poroshell 120Aq-C18柱(4.6mm×150mm，2.7μm)；流动相A为0.1％磷酸水溶液；流动相B为0.1％磷酸乙腈溶液，梯度洗脱程序见表1；流速为0.7mL/min；检测波长为210nm；柱温为30℃；进样量为5μL。Chromatographic analysis conditions: An LC-20A analysis 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 was 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.

表1梯度洗脱程序表
Table 1 Gradient elution program

计算得到当混合液中乳双歧杆菌BL-99的菌落总数3×1011cfu/mL、提取温度为4℃、提取时间为1h的菌泌素中L-蛋氨酸浓度为0.29mg/100ml、柠檬酸浓度为2.48mg/100ml。It was calculated that when the total colony count of Bifidobacterium lactis BL-99 in the mixed solution was 3×10 11 cfu/mL, the extraction temperature was 4°C, and the extraction time was 1 h, the L-methionine concentration in the secretory agent was 0.29 mg/100 ml and the citric acid concentration was 2.48 mg/100 ml.

(5)计算灭活菌体灭活前的菌落数(5) Calculate the number of colonies before inactivation of inactivated bacteria

其中，柠檬酸和L-蛋氨酸可以作为靶标物计算菌泌素提取前发酵液中的菌落数。Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

以柠檬酸作为靶标物计算菌泌素提取前发酵液中对应的菌落数时，所使用的回归方程为y＝2.289x-4.313；其中，x表示靶标物的浓度，单位为mg/100g；y表示菌落数，单位为109CFU/mL。根据计算得到的靶标物的浓度，带入回归方程，计算得到灭活菌体灭活前的菌落数。When calculating the corresponding colony count in the fermentation broth before mycobacterial extraction using citric acid as the target, the regression equation used is y = 2.289x - 4.313, where x represents the concentration of the target in mg/100g, and y represents the colony count in 109 CFU/mL. Substituting the calculated target concentration into the regression equation, the colony count before inactivation of the inactivated bacteria was calculated.

以L-蛋氨酸作为靶标物计算菌泌素提取前发酵液中对应的菌落数时，所使用的回归方程为y＝18.702x+4.464；其中，x表示靶标物的浓度，单位为mg/100g；y表示菌落数，单位为109CFU/mL。根据计算得到的靶标物的浓度，带入回归方程，计算得到灭活菌体灭活前的菌落数。
When calculating the corresponding colony count in the fermentation broth before mycobacterial extraction using L-methionine as the target, the regression equation used is y = 18.702x + 4.464; where x represents the concentration of the target in mg/100g, and y represents the colony count in 109 CFU/mL. Substituting the calculated target concentration into the regression equation, the colony count before inactivation of the inactivated bacteria was calculated.

对比例1Comparative Example 1

本实施例提供一种乳双歧杆菌BL-99发酵灭活菌，其制备方法包括以下步骤：This embodiment provides a fermentation-inactivated bacterium of Bifidobacterium lactis BL-99, the preparation method of which comprises the following steps:

1.三级种子制备，同实施例1。1. Preparation of tertiary seeds is the same as in Example 1.

2.发酵种子制备，同实施例1。2. Preparation of fermented seeds, same as in Example 1.

3.发酵液培养，同实施例1。3. Fermentation broth culture, same as Example 1.

4.菌体灭活4. Bacteria inactivation

采用无菌纯化水与所述菌泥混合进行重悬得到混合液(浓度3×1011cfu/mL)，对混合液进行热灭菌，热灭菌温度分别为70℃、80℃、90℃、100℃、121℃，时间为10min。Sterile purified water was used to mix the bacterial sludge and resuspend the mixture to obtain a mixed solution (concentration 3×10 11 cfu/mL), and the mixed solution was heat sterilized at temperatures of 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min.

测试例1：2,2-二苯基-1-苦基苯肼(DPPH)自由基清除Test Example 1: 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging

将200μL 0.2mM DPPH溶液与以200μL混合液(109cfu/mL)为原料制备的菌泌素或灭活菌混合，并在25℃避光条件下孵育30min。对照组用等体积PBS(pH 7.4)代替，空白组用等量PBS(pH 7.4)代替DPPH自由基溶液。在2,000×g离心10分钟后，测定溶液在517nm处的吸光度。计算公式如下：
Mix 200 μL of 0.2 mM DPPH solution with 200 μL of a mixture (10 9 cfu/mL) of bacteriocins or inactivated bacteria and incubate at 25°C in the dark for 30 minutes. For the control group, replace the DPPH free radical solution with an equal volume of PBS (pH 7.4). For the blank group, replace the DPPH free radical solution with an equal volume of PBS (pH 7.4). After centrifugation at 2,000 × g for 10 minutes, measure the absorbance of the solution at 517 nm. The calculation formula is as follows:

测试例2：羟自由基清除Test Example 2: Hydroxyl Radical Scavenging

向含有2.5mM 1，10-菲咯啉(1.0mL)、2.5mM FeSO4(1.0mL)和PBS(1.0mL，pH 7.4)的混合物中加入以总计1.0mL的混合液(109cfu/mL)为原料制备的菌泌素或灭活菌。添加20mM H2O2(1.0mL)后，将混合物在37℃水浴中温育90min。在536nm处获得吸光度。计算其清除羟基自由基的活性如下:
To a mixture containing 2.5 mM 1,10-phenanthroline (1.0 mL), 2.5 mM FeSO₄ (1.0 mL), and PBS (1.0 mL, pH 7.4), add 1.0 mL of the mixture ( 10⁺ cfu/mL) prepared with a secretory agent 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:

本公开中，采用LC-MS对对比例1中得到的灭活菌的成分进行检测，对检测结果中峰面积大于104的组分进行筛选得到表征灭活菌成分的靶标物，靶标物筛选标准包括：①热灭菌前不存在，或者热灭菌前该组分的含量远远低于热灭菌后该组份含量；②实验范围内，不同热灭菌条件均能够稳定存在(含量变化幅度≤20％)。发现有6种物质可以作为潜在的检测靶标物，6种物质分别为：Proly-Alanine、L-Methionine、Citric Acid、bAsp-Leu、bAsp-Phe、Antiarrhythmic peptide、GRPPK。同时，对菌泌素中6种检测靶标进行检测，发现在菌泌素中仅存在柠檬酸和L-蛋氨酸，其他四种检测靶标并未提取出来或含量低于检测线，因此本公开中菌泌素以柠檬酸和L-蛋氨酸为检测靶标表征菌泌素。In the present disclosure, LC-MS was used to detect the components of the inactivated bacteria obtained in Comparative Example 1, and the components with peak areas greater than 10 4 in the test results were screened to obtain targets for characterizing the components of the inactivated bacteria. The target screening criteria included: ① not existing before heat sterilization, or the content of the component before heat sterilization was much lower than the content of the component after heat sterilization; ② within the experimental range, different heat sterilization conditions were able to stably exist (content change range ≤ 20%). It was found that there were 6 substances that could 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 were used as detection targets to characterize the bacteriocin.

对实施例1中得到的菌泌素的羟基自由基清除能力的测试结果如图4所示，图4可以看出，当提取浓度为3×1011cfu/mL、提取温度为4℃、提取时间为1h时，菌泌素的羟基自由基清除能力较好；对实施例1中得到的菌泌素的DPPH自由基清除能力的测试结果如图5所示，图5中，K1-K9的提取温度和时间分别为4℃、1h，4℃、2h，4℃、3h，25℃、1h，25℃、2h，25℃、3h，37℃、1h，37℃、2h和37℃、3h；K10-K18的提取温度和时间分别为4℃、1h，4℃、2h，4℃、3h，25℃、1h，25℃、2h，25℃、3h，37℃、1h，37℃、2h和37℃、3h；K19-K27的提取温度和时间分别为4℃、1h，4℃、2h，4℃、3h，25℃、1h，25℃、2h，25℃、3h，37℃、1h，37℃、2h和37℃、3h。图5中，提取条件对DPPH自由基的影响相对较小。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 is 4°C, and the extraction time is 1 h, the hydroxyl radical scavenging ability of the bacteriocin is better; the test results of the DPPH radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG5 . In FIG5 , the extraction temperature and time of K1-K9 are 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; 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.

对对比例1中得到的后生元的羟基自由基清除能力和DPPH自由基清除能力如图6所示，其中菌泌素的DPPH自由基清除能力先对温度变化不大，但是121℃高温条件下明显下降。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.

所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。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.

工业实用性Industrial Applicability

本公开中的乳双歧杆菌BL-99菌泌素、乳双歧杆菌BL-99发酵灭活菌和乳双歧杆菌BL-99后生元具有抗氧化的功能，可应用于食品和药品中，且相比于包括完整死细胞+细胞壁组分+细胞膜组分+无细胞上清的混合体，本公开中的菌泌素为液体或水溶性粉末，在一些液体饮料、药品中使用更有优势。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 (19)

一种乳双歧杆菌BL-99菌泌素，其特征在于，包括乳双歧杆菌BL-99的胞吐物及乳双歧杆菌BL-99的代谢产物，所述菌泌素中柠檬酸与L-蛋氨酸质量比大于2：1，所述乳双歧杆菌BL-99的保藏编号CGMCC No.15650。A bacteriocin produced by Bifidobacterium lactis BL-99, characterized in that it comprises exocytosis products of Bifidobacterium lactis BL-99 and metabolites of Bifidobacterium lactis BL-99, the mass ratio of citric acid to L-methionine in the bacteriocin being greater than 2:1, and the preservation number of Bifidobacterium lactis BL-99 being CGMCC No. 15650. 根据权利要求1所述的乳双歧杆菌BL-99菌泌素，其特征在于，所述菌泌素中柠檬酸与L-蛋氨酸质量比为2-25：1。The Bifidobacterium lactis BL-99 secretin according to claim 1, characterized in that the mass ratio of citric acid to L-methionine in the secretin is 2-25:1. 根据权利要求1或2所述的乳双歧杆菌BL-99菌泌素，其特征在于，可选地所述菌泌素中柠檬酸与L-蛋氨酸质量比为8-9：1。The Bifidobacterium lactis BL-99 secretin according to claim 1 or 2, characterized in that, optionally, the mass ratio of citric acid to L-methionine in the secretin is 8-9:1. 一种权利要求1-3任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，包括以下步骤：A method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 1 to 3, characterized in that it comprises the following steps: 菌泌素提取，采用溶剂对发酵液中分离出的菌泥进行提取，得到混合液；Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid; 灭菌，分离出所述混合液中的上清液并进行热灭菌，得到所述菌泌素。Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin. 根据权利要求4所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，提取温度为0-37℃；可选地，提取温度为3-5℃；The method for preparing the secretin of Bifidobacterium lactis BL-99 according to claim 4, characterized in that the extraction temperature is 0-37°C; optionally, the extraction temperature is 3-5°C; 和/或，提取时间0.5-3h；可选地，提取时间30-90min。And/or, the extraction time is 0.5-3h; optionally, the extraction time is 30-90min. 根据权利要求4或5所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，所述混合液中乳双歧杆菌BL-99的菌落总数为0.5×1011-5×1011cfu/mL；可选地，所述混合液中乳双歧杆菌BL-99的菌落总数为2×1011-4×1011cfu/mL。The method for preparing a secretin from 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 is 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. 据权利要求4-6任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，热灭菌的温度为70-121℃；可选地，热灭菌的温度为70-100℃。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. 据权利要求4-7任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，热灭菌的时间为5-30min；可选地，热灭菌的时间为10-16min。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 to 30 minutes; optionally, the heat sterilization time is 10 to 16 minutes. 据权利要求4-8任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，还包括发酵步骤：将乳双歧杆菌BL-99进行液体培养，待生长到对数期时，停止发酵。The method for preparing the secretin of Bifidobacterium lactis BL-99 according to any one of claims 4 to 8 is characterized in that it further comprises a fermentation step: culturing Bifidobacterium lactis BL-99 in liquid and stopping the fermentation when the Bifidobacterium lactis BL-99 grows to the logarithmic phase. 根据权利要求4-9任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，可选地所述发酵步骤包括：将乳双歧杆菌BL-99发酵种子液接种至培养基中，先在温度30-40℃、转速60-80rpm、pH 5.8-6.2条件下发酵14-16h，得到乳双歧杆菌BL-99发酵液。The method for preparing the secretory agent of Bifidobacterium lactis BL-99 according to any one of claims 4 to 9 is characterized in that, optionally, the fermentation step comprises: inoculating the fermentation seed liquid of Bifidobacterium lactis BL-99 into the culture medium, and first fermenting it for 14 to 16 hours at a temperature of 30 to 40°C, a rotation speed of 60 to 80 rpm, and a pH of 5.8 to 6.2 to obtain the fermentation liquid of Bifidobacterium lactis BL-99. 根据权利要求4-10任意一项所述的乳双歧杆菌BL-99菌泌素的制备方法，其特征在于，可选地所述乳双歧杆菌BL-99发酵种子液的制备包括：将乳双歧杆菌BL-99菌株经活化、纯化、一级种子液培养、二级种子液扩繁、三级种子液培养和发酵种子制备，得到所述乳双歧杆菌BL-99发酵种子液；The method for preparing a secretory agent of Bifidobacterium lactis BL-99 according to any one of claims 4 to 10, characterized in that, optionally, the preparation of the fermentation seed liquid of Bifidobacterium lactis BL-99 comprises: activating the Bifidobacterium lactis BL-99 strain, purifying it, culturing it in a primary seed liquid, expanding it in a secondary seed liquid, culturing it in a tertiary seed liquid, and preparing it as a fermentation seed to obtain the fermentation seed liquid of Bifidobacterium lactis BL-99; 可选地，所述培养基为MRS液体培养基；Optionally, the culture medium is MRS liquid culture medium; 可选地，所述溶剂为水；可选地，所述溶剂为无菌水；Optionally, the solvent is water; Optionally, the solvent is sterile water; 可选地，所述灭菌步骤后，还对菌泌素进行冻干，得到冻干菌泌素。Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin. 一种乳双歧杆菌BL-99发酵灭活菌的制备方法，其特征在于，采用溶剂对发酵液中分离出的菌泥进行重悬得到混合液，对所述混合液进行热灭菌，得到所述乳双歧杆菌BL-99发酵灭活菌。A method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99 is characterized in that bacterial mud separated from fermentation broth is resuspended in a solvent to obtain a mixed liquid, and the mixed liquid is heat sterilized to obtain the fermented inactivated bacteria of Bifidobacterium lactis BL-99. 根据权利要求12所述的乳双歧杆菌BL-99发酵灭活菌的制备方法，其特征在于，所述热灭菌的温度为70-121℃，热灭菌时间为5-30min。The method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99 according to claim 12, wherein the heat sterilization temperature is 70-121°C and the heat sterilization time is 5-30 min. 根据权利要求12或13所述的乳双歧杆菌BL-99发酵灭活菌的制备方法，其特征在于，所述混合液中乳双歧杆菌BL-99的菌落总数为0.5×1011-5×1011cfu/mL。The method for preparing fermented inactivated bacteria of Bifidobacterium lactis BL-99 according to claim 12 or 13, wherein the total colony count of Bifidobacterium lactis BL-99 in the mixed solution is 0.5×10 11 -5×10 11 cfu/mL. 一种乳双歧杆菌BL-99发酵灭活菌，其特征在于，由权利要求12-14任意一项所述方法制备得到。A fermented inactivated bacterium of Bifidobacterium lactis BL-99, characterized in that it is prepared by the method according to any one of claims 12 to 14. 一种乳双歧杆菌BL-99后生元，其特征在于，包括权利要求1-3任意一项所述的乳双歧杆菌BL-99菌泌素和权利要求15所述的乳双歧杆菌BL-99发酵灭活菌中的至少一种。A Bifidobacterium lactis BL-99 postbiotic, characterized by comprising at least one of the Bifidobacterium lactis BL-99 secretin according to any one of claims 1 to 3 and the Bifidobacterium lactis BL-99 fermentation-inactivated bacteria according to claim 15. 一种权利要求16所述的乳双歧杆菌BL-99后生元在制备食品中的应用，其特征在于，所述食品包括乳制品、饮料中的至少一种。A 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 a dairy product and a beverage. 一种权利要求16所述的乳双歧杆菌BL-99后生元在制备组合物中的应用，其特征在于，所述组合物包括抗氧化组合物；优选的，所述组合物选自药物、保健食品和饲料中得至少一种。A use of the Bifidobacterium lactis BL-99 postbiotic according to claim 16 in preparing a composition, characterized in that the composition comprises an antioxidant composition; preferably, the composition is selected from at least one of a medicine, a health food and a feed. 一种权利要求16所述的乳双歧杆菌BL-99后生元在抗氧化中的应用。A use of the Bifidobacterium lactis BL-99 postbiotic according to claim 16 in anti-oxidation.
PCT/CN2024/144022 2024-02-07 2024-12-30 Bifidobacterium lactis bl-99 bacteriocin, inactivated bacteria, preparation method, and use thereof Pending WO2025167388A1 (en)

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