Bifidobacterium longum subsp. infantis b2-01 bacteriocin crude extract, and a preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and discloses a Bifidobacterium longum subsp. infantis B2-01 bacteriocin crude extract, a preparation method and application thereof, and comprises the following steps: (1) fermentation culture: Bifidobacterium longum subsp. infantis B2-01 (preservation number GDMCC No: 61911) is cultured to obtain a fermentation liquor; (2) extraction method: the fermentation liquor in step (1) is centrifuged to obtain a supernatant, the pH is adjusted to 6.0±0.5, and after freeze-drying, organic reagent extraction is carried out by adding anhydrous ethanol; the volume ratio of the supernatant to the anhydrous ethanol is 1:0.5-2.5, extraction is carried out in a 400-800 r / min magnetic stirrer for 4-8 h, and after filtration and drying, the Bifidobacterium longum subsp. infantis B2-01 bacteriocin crude extract is obtained. The bacteriocin crude extract obtained by the application has excellent stability and broad-spectrum antibacterial activity, and can be used as an antibacterial agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. More specifically, this invention relates to a method for preparing a crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin with broad-spectrum antibacterial activity and safety and non-toxicity, and its application in the preservation of fresh tremella. Background Technology

[0002] Microbial secondary metabolites are a diverse class of complex compounds synthesized by microorganisms through secondary metabolic pathways during specific growth stages or under specific environmental conditions. These compounds are not directly involved in microbial growth and reproduction, but play important roles in ecological adaptation, interspecies competition, and signal transduction. Bacteriocins, as a type of microbial secondary metabolite, are polypeptides or proteins synthesized by bacteria via ribosomes, possessing antibacterial activity and widely found in both Gram-positive and Gram-negative bacteria. Due to their natural, harmless nature and low likelihood of inducing drug resistance, they exhibit great application potential. For example, bacteriocins serve as natural preservatives, inhibiting pathogens and spoilage bacteria in food and extending shelf life. They can also be used as antibiotic alternatives to control plant and animal diseases. Furthermore, bacteriocins have inhibitory effects on multidrug-resistant bacteria and are considered potential antibiotic substitutes.

[0003] Despite some progress in the research and application of bacteriocins, the following shortcomings remain: (1) Limited antibacterial spectrum: Many bacteriocins exhibit selective antibacterial activity. For example, nisin, currently a commercially successful and widely used bacteriocin, is mainly effective against Gram-positive bacteria, with weaker inhibitory effects on Gram-negative bacteria. This limits its application in the broad-spectrum antibacterial field. (2) Stability issues: Bacteriocins are easily inactivated in complex environments (such as extreme pH, high temperature, or high salt), affecting their practical application effects. For example, PlantarumC010, a bacteriocin produced from Lactobacillus plantarum C010 isolated from cow dung, and Plantaricin JY22, a bacteriocin produced from Lactobacillus plantarum JY22 isolated from golden carp intestines, are completely inactivated at pH ≥ 6, with a residual activity of 0%. (3) Safety issues: Bacteriocins are widely sourced, but not all of them have food-grade safety, and while inhibiting harmful bacteria, they also pose a risk of reducing beneficial bacteria. For example, the enterococcal Cr17 disclosed in ZL 201811611740.3 is derived from Enterococcus faecalis Gr17, which belongs to the Enterococcus genus. Because it is usually regarded as a potential indicator bacterium or pathogen, it is not included in my country's "List of Microbial Strains that can be Used in Food". Moreover, enterococcal Cr17 has a strong antibacterial effect on six strains of Bacillus subtilis. (4) Production cost issues: The production process of bacteriocins is extremely complex, involving multiple steps such as fermentation, crude extraction, preliminary purification and fine purification, and it is only at the laboratory research stage. For example, the production of Lactobacillus sakei ZFM225 bacteriocin disclosed in ZL 202210476564.7 includes six steps: fermentation, ammonium sulfate precipitation, desalting, cation exchange chromatography, secondary desalting and reversed-phase high-performance liquid chromatography purification. The process is complex and the production cost is high.

[0004] Bifidobacterium longum, belonging to the genus Bifidobacterium, is a commensal bacterium in the human gastrointestinal tract. It is Gram-positive, anaerobic, produces lactic acid, does not form sporulations, and is non-motile. This species includes three subspecies: *Bifidobacterium longum* ssp. *infantis*, *Bifidobacterium longum* ssp. *longum*, and *Bifidobacterium longum* ssp. *suis* and *Bifidobacterium longum* ssp. *suillum*. *Bifidobacterium longum* and *Bifidobacterium longum* ssp. *infantis* have been validated by extensive clinical trials and are considered to have edible safety levels, both listed in my country's "List of Microbial Strains that Can Be Used in Food." However, no broad-spectrum antibacterial bacteriocins have yet been discovered in *Bifidobacterium longum*.

[0005] Therefore, there is an urgent need to find hydrophilic bacteriocins with broad-spectrum antibacterial activity, high stability, low production cost, and high safety. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a crude extract of a bacteriocin from Bifidobacterium longum subsp. infantis B2-01.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The method for preparing the crude bacteriocin extract of Bifidobacterium longum subsp. infantis B2-01 includes the following steps:

[0009] (1) Fermentation culture: 2% to 5% of Bifidobacterium longum subsp. infantis B2-01 was inoculated into MRS liquid medium and cultured at 30 to 41°C for 32 to 40 hours to obtain fermentation broth; the pH of the MRS liquid medium was adjusted to 6.0 to 7.0 before sterilization; the preservation number of Bifidobacterium longum subsp. infantis B2-01 is GDMCC No:61911;

[0010] (2) Extraction method: The fermentation broth from step (1) was centrifuged to obtain the supernatant, and the pH was adjusted to 6.0±0.5; after freeze-drying, anhydrous ethanol was added for organic reagent extraction. The volume ratio of the supernatant to anhydrous ethanol was 1:0.5~2.5. The extraction was carried out at 400~800r / min with a magnetic stirrer for 4~8h. After filtration and drying, the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin was obtained.

[0011] Preferably, the inoculation amount in step (1) is 3.5±0.5%, the culture temperature is 36±2℃, and the culture time is 36±4h.

[0012] Preferably, the formulation of the MRS liquid culture medium is as follows: 2±1 parts glucose, 1±0.5 parts beef extract powder, 1±0.5 parts casein digest, 0.4±0.2 parts yeast extract powder, 0.5±0.25 parts sodium acetate, 0.2±0.1 parts triammonium citrate, 0.2±0.1 parts dipotassium hydrogen phosphate, 0.108±0.05 parts Tween-80, 0.02±0.01 parts magnesium sulfate heptahydrate, 0.005±0.002 parts manganese sulfate tetrahydrate, 0.005±0.002 parts ascorbic acid, and 0.005±0.002 parts L-cysteine ​​hydrochloride, diluted to 100 parts with distilled water, and the pH is adjusted to 6.5±0.2.

[0013] Preferably, the centrifugation conditions in step (2) are 2-6℃, 6000-10000r / min for 3-7min.

[0014] Preferably, the volume ratio of the supernatant to anhydrous ethanol is 1:1±0.5.

[0015] Preferably, the Bifidobacterium longum subsp. infantis B2-01 undergoes two activation cultures before inoculation, with the activation culture temperature being 37±2℃ and the culture time being 16±2h.

[0016] Preferably, the drying process involves rotary evaporating the filtrate at 40–50°C until it is completely dried.

[0017] The aforementioned *Bifidobacterium longum* subsp. *infantitidis* B2-01 and its fermentation supernatant and crude bacteriocin extract have any of the following uses:

[0018] (1) Use in products that inhibit the growth of putrefactive or pathogenic bacteria to extend shelf life, including feed, cosmetics, pharmaceuticals, food and other fields.

[0019] (2) In the food industry, it has the potential to be applied to foods that require high-temperature treatment, need to coexist with colorants, are in high-salt environments, or are slightly acidic environments, and can have a synergistic effect with ultraviolet irradiation.

[0020] (3) Provide novel alternative antibiotic solutions for contamination caused by microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Enterobacter sakazakii, Salmonella typhimurium, Pseudomonas aeruginosa, Shewanella putrefactive bacteria, Pseudomonas fluorescens, Burkholderia calcei, Penicillium, Rhizopus, Aspergillus flavus, Monascus purpureus, and Candida albicans.

[0021] Preferably, the application of the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocinolone in food preservation.

[0022] Preferably, the crude extract is sprayed onto fresh white fungus after surface impurities have been removed and allowed to be naturally absorbed and air-dried. This can alleviate the increase in weight loss, decrease in moisture content, decrease in Hunter whiteness, increase in total bacterial count, and decrease in total soluble sugar content of fresh white fungus during storage, thereby reducing the rate of loss of the commercial value of fresh white fungus.

[0023] Preferably, the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin is used in the preparation of antibacterial agents.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The crude bacteriocin extract of Bifidobacterium longum subsp. infantis B2-01 prepared by this invention has high antibacterial activity against a variety of microorganisms, including Gram-negative and Gram-positive bacteria, and has a broad antibacterial spectrum. At the same time, the crude bacteriocin extract of this invention has no antibacterial activity against probiotics such as Lactobacillus rhamnosus and Lactobacillus plantarum.

[0026] (2) The crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocins prepared by the present invention has good stability, including UV irradiation stability, thermal stability, high salt stability, surfactant stability, metal ion stability and pH stability.

[0027] (3) The crude bacteriocin extract of Bifidobacterium longum subsp. infantis B2-01 prepared by this invention has high safety. The Bifidobacterium longum subsp. infantis B2-01 strain is one of the strains listed in my country's "List of Microbial Strains that Can Be Used in Food", and the crude bacteriocin extract produced by it has high safety.

[0028] (3) This invention simplifies the production process of bacteriocins to two steps: fermentation and crude extraction. It replaces the ammonium sulfate precipitation method with organic reagent extraction, eliminating the desalting step. The crude bacteriocin extract obtained also has high antibacterial activity and application value. This invention greatly reduces production costs and is conducive to the industrial production of bacteriocins.

[0029] (4) The crude extract of Bifidobacterium longum bacteriocins obtained by the present invention has excellent stability and broad-spectrum antibacterial activity, and can be used as an antibacterial agent and for antibacterial preservation of various products.

[0030] (5) This invention discloses for the first time the bacteriocin gene cluster sequence of Bifidobacterium longum infant subspecies B2-01. By using secondary metabolite mining tools such as antiSMASH and database annotation information, the similarity with other bacteriocin gene clusters is less than 40%, indicating that Bifidobacterium longum infant subspecies B2-01 produces a new bacteriocin.

[0031] This invention is based on the strain Bifidobacterium longum subsp. infantis B2-01 disclosed in ZL 202210850462.7. The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) on September 1, 2021, with accession number GDMCC No:61911. Attached Figure Description

[0032] Figure 1 This is a diagram of the gene clusters annotated by antiSMASH in Bifidobacterium longum B2-01 in Example 3.

[0033] Figure 2 The figures show the growth curve, pH change curve, and antibacterial effect change of Bifidobacterium longum B2-01 in Example 4.

[0034] Figure 3 shows the effects of different single factors on bacteriocin production by *Bifidobacterium longum* subsp. *infantii* B2-01 in Example 4. (a) shows the effect of different inoculation ages on bacteriocin production by *Bifidobacterium longum* subsp. *infantii* B2-01; (b) shows the effect of different culture temperatures on bacteriocin production by *Bifidobacterium longum* subsp. *infantii* B2-01; (c) shows the effect of different initial pH values ​​on bacteriocin production by *Bifidobacterium longum* subsp. *infantii* B2-01; and (d) shows the effect of different inoculation amounts on bacteriocin production by *Bifidobacterium longum* subsp. *infantii* B2-01. Different lowercase letters in each figure indicate significant differences between groups (p < 0.05), and the same applies below.

[0035] Figure 4 shows the response surface and contour plots of the interaction of various factors in the Box-Behnken experiment in Example 4; where (a) and (b) are the response surface and contour plots of initial pH and inoculum size; (c) and (d) are the response surface and contour plots of initial pH and culture time; and (e) and (f) are the response surface and contour plots of inoculum size and culture time.

[0036] Figure 5 shows the effects of various crude extraction methods in Example 5 on the bacteriocin activity and yield of *Bifidobacterium longum* subsp. *infantii* B2-01. (a) is the standard curve of antibacterial potency; (b) is the phase diagram of anhydrous ethanol / ammonium sulfate aqueous two-phase system; (c) shows the antibacterial activity of the crude extract at different ammonium sulfate saturations; (d) shows the effect of different concentrations of anhydrous ethanol on the yield of the antibacterial active substance; (e) shows the effect of different concentrations of ammonium sulfate on the yield of the antibacterial active substance; (f) shows the effect of different water-insoluble organic reagents on the yield of the antibacterial active substance; and (g) shows the effect of different extraction ratios of supernatant to n-butanol on the yield of the antibacterial active substance.

[0037] Figure 6 shows the effects of various factors on the antibacterial activity stability of the crude extract of *Bifidobacterium longum* subsp. *infantitidis* B2-01 bacteriocin in Example 6. Specifically, (a) shows the effect of different UV irradiation times on the antibacterial activity of the crude extract; (b) shows the effect of different temperature treatments for 30 min on the antibacterial activity of the crude extract; (c) shows the effect of different mass fractions of NaCl treatment on the antibacterial activity of the crude extract; (d) shows the effect of different surfactant treatments with a volume fraction of 10% on the antibacterial activity of the crude extract; (e) shows the effect of different metal ion treatments with an ion concentration of 5 mmol / L on the antibacterial activity of the crude extract; and (f) shows the effect of different pH values ​​on the antibacterial activity of the crude extract.

[0038] Figure 7 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) of the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocinolone against Burkholderia gladioli CICC 10574 were determined in Example 8.

[0039] Figure 8 shows the effects of different treatment methods on the quality changes of fresh tremella during 20 days of storage at 4℃ in Example 8. Among them, (a) is the effect of different treatment methods on the weight loss rate of fresh tremella during storage; (b) is the effect of different treatment methods on the total bacterial count of fresh tremella during storage; and (c) is the effect of different treatment methods on the Hunter whiteness of fresh tremella during storage. Detailed Implementation

[0040] The following specific embodiments are intended to illustrate the present invention in detail for better understanding, and are not intended to limit its scope of protection.

[0041] Unless otherwise specified in the embodiments, the process parameters, experimental steps or conditions can be operated in accordance with conventional techniques in the field or general experimental methods in relevant literature; reagents or instruments that do not specify a specific manufacturer are all conventional products that can be obtained through conventional commercial channels.

[0042] Example 1: Fermentation method of Bifidobacterium longum subsp. infantis B2-01

[0043] This embodiment provides a fermentation method for Bifidobacterium longum subsp. infantis B2-01.

[0044] (1) Preparation of Bifidobacterium longum B2-01 seed culture

[0045] Take 2-4% (v / v) of Bifidobacterium longum subsp. infantis B2-01 from a skim milk cryopreservation tube stored at -80℃ and inoculate it into a specially prepared MRS liquid medium that has been autoclaved at 121℃ for 15-20 min and then cooled to 40-45℃. Then, incubate it in a 37℃ constant temperature anaerobic incubator for 16-18 h and activate it twice to obtain the seed culture.

[0046] (2) Preparation of fermentation supernatant of Bifidobacterium longum B2-01

[0047] Take the activated Bifidobacterium longum B2-01 seed culture and inoculate it at a ratio of 3% (v / v) into a specially prepared MRS liquid culture medium that has been sterilized at 121℃ for 15-20 min and then cooled to 40-45℃. After incubating in a 37℃ constant temperature anaerobic incubator for 18 h, centrifuge at 4℃ and 8000 r / min for 5 min to obtain the supernatant for later use.

[0048] The formula for the specially prepared MRS liquid culture medium is as follows: 2 parts glucose, 1 part beef extract powder, 1 part casein digest, 0.4 parts yeast extract powder, 0.5 parts sodium acetate, 0.2 parts triammonium citrate, 0.2 parts dipotassium hydrogen phosphate, 0.108 parts Tween-80, 0.02 parts magnesium sulfate heptahydrate, 0.005 parts manganese sulfate tetrahydrate, 0.005 parts ascorbic acid, 0.005 parts L-cysteine ​​hydrochloride, and distilled water to a final volume of 100 parts. The pH is adjusted to 7.00±0.02 before sterilization.

[0049] Example 2: Preliminary confirmation of bacteriocin production by Bifidobacterium longum subsp. infantis B2-01

[0050] This embodiment provides preliminary confirmation that bacteriocins are present in the fermentation supernatant obtained in Example 1. Specifically, after excluding interference from bacterial cells, organic acids, and hydrogen peroxide, the fermentation supernatant was determined to have antibacterial activity.

[0051] (1) Elimination of bacterial cell interference

[0052] The fermentation supernatant was sterilized by filtration through a 0.22 μm PES filter membrane, and the antibacterial activity was measured using unfiltered fermentation supernatant as a control.

[0053] (2) Elimination of interference from organic acids

[0054] The pH of the fermentation supernatant was adjusted to 6.00 ± 0.02 with 1 mol / L NaOH solution and then filtered through a 0.22 μm PES membrane for sterilization. The unadjusted fermentation supernatant served as a blank control. Sterile distilled water was adjusted to pH 6.00 ± 0.02 with lactic acid and acetic acid respectively as negative controls, and then filtered through a 0.22 μm PES membrane for sterilization before its antibacterial activity was measured.

[0055] (3) Elimination of hydrogen peroxide interference

[0056] Catalase was dissolved in PBS buffer beforehand. Fermentation supernatant with pH = 6.00 ± 0.02 was taken, and catalase was added to make a final concentration of 1 mg / mL. After incubation at 37°C for 2 h, the mixture was filtered through a 0.22 μm PES membrane for sterilization. An equal volume of PBS buffer was added to fermentation supernatant with pH = 6.00 ± 0.02 that was not treated with catalase as a control. The mixture was then filtered through a 0.22 μm PES membrane for sterilization, and the antibacterial activity was determined.

[0057] The antibacterial activity determination is performed by using the agar diffusion method to obtain the average diameter of the inhibition zone and evaluating the antibacterial activity of the sample to be tested. The method is as follows:

[0058] (1) Activation culture of indicator bacteria

[0059] Indicator bacteria Escherichia coli ATCC 25922 and Staphylococcus aureus RN 4220 were taken from glycerol cryovials stored at -80°C and inoculated into sterilized LB liquid medium at a ratio of 1% (v / v). The medium was then incubated at 37°C and 180 rpm for 12–18 h with shaking, and activated twice for later use.

[0060] (2) Determination of antibacterial activity

[0061] Add activated 1×10⁻⁶ agar to LB solid medium that has been sterilized and cooled to 45–55°C and contains 0.75–1% agar. 5 Inhibition indicator bacteria at CFU / mL were prepared by shaking and then pouring the plate to solidify, thus obtaining an inhibition plate. Holes were punched in the inhibition plate using a 9mm outer diameter punch, and 100μL of the sample to be tested was added to each hole. After diffusion, the plate was incubated at 37℃ for 8 hours. The plate was then removed for observation, and the diameter of the inhibition zone was measured. Three replicates were set for each group.

[0062] The results are shown in Table 1. After removing bacterial cells from the fermentation supernatant, the diameter of the inhibition zone was not significantly different from that of the untreated original fermentation broth, indicating that the antibacterial activity of the fermentation supernatant was not produced by bacterial cells. The diameter of the inhibition zone of the fermentation supernatant with adjusted pH = 6.00 ± 0.02 was significantly smaller than that of the untreated fermentation supernatant. This is because the untreated original fermentation supernatant contained a large amount of organic acids such as lactic acid and acetic acid produced by the culture of Bifidobacterium longum B2-01, which had a strong inhibitory effect on indicator bacteria. After excluding the influence of organic acids, although the antibacterial activity of the fermentation supernatant decreased to some extent, neither lactic acid nor acetic acid at pH = 6.00 ± 0.02 showed any antibacterial activity, indicating that the antibacterial activity of the fermentation supernatant after adjusting pH = 6.00 ± 0.02 was not due to the effect of organic acids. Meanwhile, the antibacterial activity of the fermentation supernatant treated with catalase was not significantly different from that of the untreated broth, indicating that the antibacterial activity of the fermentation supernatant was not due to the effect of hydrogen peroxide. Therefore, the fermentation supernatant of Bifidobacterium longum B2-01 also contains bacteriocins in addition to the above-mentioned antibacterial substances.

[0063] Table 1. Effects of different treatments on the antibacterial activity of fermentation supernatant

[0064]

[0065]

[0066] Note: Different lowercase letters in the table indicate significant differences between groups (p < 0.05), and the same applies below.

[0067] Example 3: Mining of the bacteriocin gene cluster of Bifidobacterium longum subsp. infantis B2-01

[0068] In this embodiment, the genome of Bifidobacterium longum B2-01 was sequenced by combining second-generation Illumina and third-generation ONT technologies. The gene cluster sequence encoding secondary metabolite synthesis was mined by the secondary metabolite mining tool antiSMASH in combination with database annotation information to mine its bacteriocinogen gene cluster.

[0069] The results are as follows Figure 1As shown, a total of five predicted bacteriocin gene clusters were obtained: two clusters encoding RiPP (ribosomal synthesized and post-translational modified peptides) (Cluster 2, Cluster 5), one cluster encoding NRPS (non-ribosomal polypeptide synthase) (Cluster 1), one cluster encoding class I lanothiopeptide (Cluster 3), and one cluster encoding class IV lanothiopeptide (Cluster 4). The similarity of these five gene clusters was all below 40%, indicating that they are bacteriocin gene clusters unique to *Bifidobacterium longum* B2-01, and the bacteriocins translated and expressed by them are novel bacteriocins.

[0070] Example 4: Optimization of Antibacterial Fermentation Culture Conditions for Bifidobacterium longum Subsp. Infantii B2-01

[0071] This embodiment uses a specially formulated MRS liquid medium as the basal medium and antibacterial activity against E. coli ATCC 25922 as the indicator. Single-factor experiments combined with Plackett-Burman and Box-Behnken response surface methodology were used to optimize the culture conditions, including inoculation time, inoculation age, culture temperature, initial pH, and inoculation amount. The formulation of the specially formulated MRS liquid medium is the same as in Example 1, and the determination of antibacterial activity is the same as in Example 2.

[0072] (1) Single-factor experiment and corresponding results

[0073] In the single-factor experiment of culture time, the activated Bifidobacterium longum B2-01 seed culture from Example 1 was inoculated into sterilized special MRS liquid medium at a ratio of 3% (v / v) and cultured in a constant temperature anaerobic incubator at 37℃. The OD of the bacterial culture was measured every 4 hours. 600nm After adjusting the pH, centrifuge at 4℃ and 8000 r / min for 5 min, take the supernatant, adjust the pH to 6.00±0.02, filter through a 0.22 μm PES membrane for sterilization, and then determine the antibacterial activity using the agar diffusion method. Results are as follows: Figure 2 As shown in the bar chart, the relationship between the growth time of Bifidobacterium longum B2-01 and the antibacterial activity of its fermentation supernatant is as follows: The fermentation supernatant showed no antibacterial activity from 0-8 h. From 12 h onwards, the fermentation supernatant exhibited certain antibacterial activity, which was positively correlated with time. The antibacterial activity was strongest at 36 h, with an inhibition zone diameter of 18.47 ± 0.21 mm (P < 0.05). After 36 h, the antibacterial activity of the fermentation supernatant showed a negative correlation with time. This is likely due to the increased number of dead bacterial cells in the later stages of culture, leading to the outflow of intracellular substances and affecting the antibacterial activity of the bacteriocins accumulated in the fermentation supernatant to some extent.

[0074] In the single-factor experiment on inoculation age, *Bifidobacterium longum* B2-01 seed culture with secondary activation culture times of 10h, 12h, 14h, 16h, 18h, and 20h were inoculated into sterilized specially prepared MRS liquid medium at a ratio of 3% (v / v). After incubation in a constant temperature anaerobic incubator at 37℃ for 18h, the culture was centrifuged at 4℃ and 8000r / min for 5min. The supernatant was collected, the pH was adjusted to 6.00±0.02, and the culture was filtered through a 0.22μm PES filter for sterilization before the antibacterial activity was measured. The results are shown in Figure 3(a). The inhibition zone diameter was the largest when the inoculation age was 16h, reaching 19.47±0.17mm (P<0.05). Therefore, 16h was selected as the optimal inoculation age for subsequent studies.

[0075] In the single-factor experiment of culture temperature, the activated Bifidobacterium longum B2-01 seed culture from Example 1 was inoculated into sterilized specially prepared MRS liquid medium at a ratio of 3% (v / v). The culture was incubated in constant temperature anaerobic incubators at 25℃, 30℃, 37℃, 41℃, and 45℃ for 18 h, then centrifuged at 4℃ and 8000 r / min for 5 min. The supernatant was adjusted to pH 6.00±0.02, filtered through a 0.22 μm PES filter for sterilization, and the antibacterial activity was measured. The results are shown in Figure 3(b). The inhibition zone diameter was largest at a culture temperature of 37℃, reaching 19.18±0.16 mm (P<0.05). Therefore, 37℃ was selected as the optimal culture temperature for subsequent studies.

[0076] In the single-factor experiment on initial pH, sterilized specially prepared MRS liquid culture medium was used, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 using 1 mol / L NaOH and 1 mol / L HCl solutions. The activated Bifidobacterium longum B2-01 seed culture from Example 1 was inoculated at a ratio of 3% (v / v), and cultured in a 37℃ anaerobic incubator for 18 h. After centrifugation at 4℃ and 8000 r / min for 5 min, the supernatant was collected and the pH was adjusted to 6.00 ± 0.02. The culture was then filtered through a 0.22 μm PES membrane for sterilization before the antibacterial activity was measured. The results are shown in Figure 3(c). The inhibition zone diameter was largest at an initial pH of 6.5, reaching 18.33 ± 0.12 mm (P < 0.05). Therefore, pH = 6.5 was selected as the optimal initial pH for subsequent studies.

[0077] In the single-factor experiment on inoculum size, the activated Bifidobacterium longum B2-01 seed culture from Example 1 was inoculated into sterilized specially prepared MRS liquid medium at proportions of 0.5%, 1%, 2%, 3%, 5%, and 10% (v / v). After incubation in a constant temperature anaerobic incubator at 37℃ for 18 h, the medium was centrifuged at 4℃ and 8000 r / min for 5 min. The supernatant was taken, the pH was adjusted to 6.00±0.02, and the medium was filtered through a 0.22 μm PES filter for sterilization before the antibacterial activity was measured. The results are shown in Figure 3(d). The inhibition zone diameter was the largest when the inoculum size was 3.0%, reaching 19.20±0.22 mm (P<0.05). Therefore, 3.0% was selected as the optimal inoculum size for subsequent studies.

[0078] (2) Plackett-Burman test to screen for significant influencing factors

[0079] A Plackett-Burman experimental design was used to screen the factors that most significantly affected the antibacterial activity of Bifidobacterium longum subsp. infantis B2-01 supernatant, considering single-factor inoculation time, inoculation age, culture temperature, initial pH, and inoculation amount. The baseline value of "0" was set at the point of maximum antibacterial activity for all five factors. Two levels were selected: a low level and a high level. The low-level response was converted to a code value of "-1", and the high-level response was converted to a code value of "1". The experimental design and results are shown in Table 2.

[0080] Table 2. Plackett-Burman Experimental Design and Results

[0081]

[0082] Table 3 shows the effect analysis of the Plackett-Burman experiment. It can be seen that the effects of inoculation age, culture temperature, and initial pH are negative, therefore the amounts of these three factors need to be reduced. Conversely, the effects of culture time and inoculation amount are positive, requiring an increase in these three factors. The contribution rate of each factor indicates its importance to the model. The contribution rates of different factors, from highest to lowest, are: initial pH > inoculation amount > culture time > inoculation age > culture temperature. Therefore, initial pH, inoculation amount, and culture time were selected for the Box-Behnken response surface design. The overall factor model P = 0.0380 < 0.05, indicating that the results of the Plackett-Burman experiment are reliable.

[0083] Table 3. Analysis of the effects of the Plackett-Burman experiment.

[0084]

[0085] (3) Box-Behnken response surface experimental design and verification

[0086] A three-factor, three-level Box-Behnken experiment was designed using Design-Expert 13 software to determine the initial pH of the culture medium, the inoculum size, and the culture time. The experimental design and results are shown in Table 4.

[0087] Table 4. Box-Behnken response surface experimental design and results

[0088]

[0089] The results of the regression model analysis of variance are shown in Table 5. The regression equation encoding the inhibition zone diameter (Y) on the initial pH value (A), inoculum size (B), and culture time (C) is: Y = 19.55 + 0.1163A - 0.1950B + 0.1038C - 0.0350AB + 0.0325AC + 0.0750BC - 2.06A 2 -2.13B 2 -1.54C 2 The model's P < 0.0001 indicates that the overall model is significant, while the lack-of-fit term P = 0.0551 > 0.05 indicates that the lack-of-fit term is not significant, and the model selection is appropriate. The model's coefficient of determination R0 is... 2 =0.9867, indicating that the model's predicted values ​​fit the actual values ​​well, and the correction determination coefficient is 0.9696, indicating that the model has high reliability.

[0090] Table 5. Results of ANOVA for the Regression Model

[0091]

[0092] The response surfaces and contour plots of the pairwise interactions of each influencing factor in the experimental design are shown in Figure 4. Using Design-Expert 13 analysis, the culture conditions with the highest antibacterial activity in the fermentation supernatant were an initial pH of 6.51, an inoculum size of 3.43%, and a culture time of 36.13 h, at which the inhibition zone diameter was the largest at 19.56 mm. Based on the above optimizations and actual conditions, three antibacterial experiments were conducted with an initial pH of 6.50, an inoculum size of 3.5%, and a culture time of 36 h to further verify the optimization results. The verification results showed that the diameter of the inhibition zone in the fermentation supernatant of *Bifidobacterium longum* B2-01 after optimizing the culture conditions was 19.26 ± 0.13 mm, which, after subtracting the pore size of the punch, was 1.37 times that before optimization, consistent with the predicted value, and the fitting rate reached 99.49%.

[0093] Example 5

[0094] The *Bifidobacterium longum* B2-01 seed culture, which underwent secondary activation and culture for 18 hours in Example 1, was inoculated at a ratio of 5% (v / v) into a sterile, specially prepared MRS liquid medium. The pH of the medium was adjusted to 6.0 using 1 mol / L NaOH and 1 mol / L HCl solutions. After incubation at 30°C for 32 hours in an anaerobic incubator, the medium was centrifuged at 4°C and 8000 rpm for 5 minutes. The supernatant was collected, and the pH was adjusted to 6.00 ± 0.02. The supernatant was then filtered through a 0.22 μm PES membrane for sterilization before its antibacterial activity was measured. The formulation of the specially prepared MRS liquid medium was the same as in Example 1, and the determination of its antibacterial activity was the same as in Example 2. The diameter of the inhibition zone in the fermentation supernatant of *Bifidobacterium longum* B2-01 after optimization of culture conditions was 18.67 ± 0.12 mm, which, after subtracting the pore size of the punch, was 1.29 times that before optimization.

[0095] Example 6

[0096] The *Bifidobacterium longum* B2-01 seed culture, which underwent secondary activation and culture for 18 hours in Example 1, was inoculated at a ratio of 5% (v / v) into a sterile, specially prepared MRS liquid medium. The pH of the medium was adjusted to 6.0 using 1 mol / L NaOH and 1 mol / L HCl solutions. After incubation at 41°C for 40 hours in an anaerobic incubator, the medium was centrifuged at 4°C and 8000 rpm for 5 minutes. The supernatant was collected, and the pH was adjusted to 6.00 ± 0.02. The supernatant was then filtered through a 0.22 μm PES membrane for sterilization before its antibacterial activity was measured. The formulation of the specially prepared MRS liquid medium was the same as in Example 1, and the determination of its antibacterial activity was the same as in Example 2. The diameter of the inhibition zone in the fermentation supernatant of *Bifidobacterium longum* B2-01 after optimization was 17.90 ± 0.09 mm, which, after subtracting the pore size of the perforator, was 1.19 times that before optimization.

[0097] Example 7: Investigation of a crude extraction method for bacteriocins produced by *Bifidobacterium longum* subsp. *infantii* B2-01

[0098] This embodiment, based on a specially prepared MRS liquid culture medium and the optimal culture conditions in Example 4, explores the optimal crude extraction method for Bifidobacterium longum B2-01 bacteriocin by comparing ammonium sulfate fractionation precipitation, aqueous two-phase extraction, and organic reagent extraction, and considering practical considerations. The antibacterial activity was determined using E. coli ATCC 25922 as the indicator bacterium, following the same method as in Example 2.

[0099] (1) Ammonium sulfate fractionation precipitation method

[0100] A certain amount of fermentation supernatant with pH = 6.00 ± 0.02 was taken, and the ammonium sulfate saturation in the fermentation supernatant was adjusted to 20% according to the commonly used ammonium sulfate saturation table. After magnetic stirring at 600 r / min for 12 h, it was centrifuged at 8000 r / min for 5 min at 4℃. The precipitate obtained by centrifugation was dissolved in distilled water and temporarily stored at 4℃. The supernatant was then used for the next stage of precipitation. The ammonium sulfate saturation was adjusted to 30%, and the above method was used to obtain precipitates and temporarily store them. This process was repeated until the ammonium sulfate saturation of the fermentation supernatant was 40%, 50%, 60%, 70%, 80%, and 90%, and then the antibacterial activity was measured. The results are shown in Figure 5(c). When the ammonium sulfate saturation was 60%, the crude extract had the strongest antibacterial activity, which was significantly higher than that of the crude extract precipitated at other saturations. The specific activity of the crude extract at this saturation reached 130.75 AU / mg.

[0101] (2) Anhydrous ethanol / ammonium sulfate aqueous two-phase extraction method

[0102] Anhydrous ethanol was added dropwise to an ammonium sulfate solution of a certain concentration. When the solution became turbid, the mass fractions of anhydrous ethanol and ammonium sulfate were recorded. A small amount of distilled water was added to clarify the solution. Anhydrous ethanol was added dropwise again, and the above operation was repeated until the mass fraction of ammonium sulfate was below 10%. Anhydrous ethanol / ammonium sulfate aqueous two-phase phase diagram 5(b) was plotted with the mass fraction of ammonium sulfate as the abscissa and the mass fraction of anhydrous ethanol as the ordinate. The area above the curve represents the two-phase region. At this concentration, the system consists of two immiscible phases: the upper phase is the anhydrous ethanol phase, and the lower phase is the ammonium sulfate phase. Therefore, the mass fractions of anhydrous ethanol and ammonium sulfate above the curve were selected for subsequent experiments.

[0103] To investigate the effect of anhydrous ethanol concentration on the antibacterial activity of crude extract, a certain amount of fermentation supernatant with pH = 6.00 ± 0.02 was taken, and ammonium sulfate was added to make its mass fraction 20%. Anhydrous ethanol was then added to make its mass fractions 30%, 32%, 34%, 36%, 38%, 40%, 42%, and 44%, respectively. After thorough stirring and extraction for 30 min, the mixture was allowed to stand at 4℃ for 6 h for phase separation. The volumes of the upper and lower phases were recorded, and the antibacterial activity of the upper and lower phase solutions was measured. The extraction rate and partition coefficient of each group were calculated. The results are shown in Figure 5(d). The partition coefficient of each experimental group was greater than 1, indicating that the bacteriocin of Bifidobacterium longum B2-01 was mainly found in the upper phase of anhydrous ethanol. Among them, the extraction rate and partition coefficient showed a trend of first increasing and then decreasing with the increase of anhydrous ethanol mass fraction. When the mass fraction of anhydrous ethanol was 38% and 40%, the extraction rate was higher than 98% and there was no significant difference. However, the partition coefficient of anhydrous ethanol mass fraction of 38% was higher than that of anhydrous ethanol mass fraction of 40%, indicating that the upper phase of the aqueous two-phase mixture formed by anhydrous ethanol mass fraction of 38% and ammonium sulfate contained a higher amount of antibacterial active substances. Therefore, anhydrous ethanol mass fraction of 38% was selected for further research.

[0104] In investigating the effect of ammonium sulfate concentration on the antibacterial activity of the crude extract, the mass fraction of anhydrous ethanol in the system was adjusted to 38%, and ammonium sulfate was added to achieve mass fractions of 22%, 24%, 26%, 28%, 30%, and 32%, respectively. Subsequent steps were the same as above. The results are shown in Figure 5(e). With the increase of ammonium sulfate mass fraction, both the extraction rate and the partition coefficient showed a trend of first increasing and then decreasing. When the mass fraction of ammonium sulfate was 26% and 28%, the extraction rate was higher than 98% and there was no significant difference. However, the partition coefficient at a mass fraction of 28% ammonium sulfate was higher than that at a mass fraction of 26%, indicating that the upper phase of the aqueous two-phase mixture formed by ammonium sulfate and anhydrous ethanol at a mass fraction of 28% contained a higher amount of antibacterial active substances, at which point the extraction rate reached 99.58%.

[0105] (3) Organic reagent extraction method

[0106] In investigating the crude extraction of antibacterial substances using water-insoluble organic reagents as extractants, a certain amount of fermentation supernatant with pH = 6.00 ± 0.02 was taken, and chloroform, ethyl acetate, n-butanol, petroleum ether, and n-hexane were added in equal volumes multiple times as extractants. Each extraction was carried out for 30 min with a magnetic stirrer at 600 r / min. The organic phases were then combined, evaporated at 45℃ until completely dry, and then reconstituted with 0.05 times the original volume of distilled water. The antibacterial activity of the reconstituted solution was measured, and the partition coefficient and extraction rate were calculated. The results are shown in Figure 5(f). The crude extract with chloroform showed no antibacterial activity; although the crude extract with ethyl acetate showed some antibacterial activity, its extraction rate and partition coefficient were too low. Neither of these were the most effective extractants for Bifidobacterium longum B2-01 bacteriocins. The antibacterial activities of the crude extracts from the other three organic reagents were: n-butanol > petroleum ether > n-hexane. n-Butanol had the highest extraction rate and partition coefficient, at 75.26% and 3.04, respectively. Therefore, n-Butanol was chosen to study the effect of different extraction ratios on the yield of antibacterial active substances.

[0107] In investigating the effect of extraction ratio on the antibacterial activity of crude extract, n-butanol was used as a water-insoluble organic reagent. The volume ratio of n-butanol to the original fermentation supernatant was changed to 0.5:1, 1:1, 1.5:1, 2:1, and 2.5:1. The extraction steps were repeated, and the antibacterial activity of the reconstituted solution was measured to calculate the specific activity. The results are shown in Figure 5(g). The antibacterial activity of the crude extract was positively correlated with the amount of n-butanol used. However, with the increase of n-butanol usage, the specific activity of the crude extract showed a trend of first increasing and then decreasing. This indicates that while increasing the amount of n-butanol allows for the extraction of more antibacterial active substances to a certain extent, it also extracts more impurities other than antibacterial active substances. When the ratio of fermentation supernatant to n-butanol was 1:1, the specific activity of the crude extract reached a maximum of 265.56 AU / mg.

[0108] In investigating the crude extraction of antibacterial substances using water-soluble organic reagents as extractants, a certain amount of fermentation supernatant with pH = 6.00 ± 0.02 was taken, freeze-dried, and then extracted with methanol and anhydrous ethanol as extractants in equal volumes. The mixture was then extracted with a magnetic stirrer at 600 r / min for 6 h. After filtration, the filtrate was rotary evaporated at 45℃ until completely dry, and then reconstituted with 0.05 times the original volume of distilled water. The antibacterial activity of the reconstituted solution was measured, and the specific activity was calculated. The results are shown in Table 6. When extracting from the fermentation supernatant with equal volumes of organic reagents, the antibacterial activity of the crude extracts from each organic reagent was: ethanol > methanol > n-butanol, with the ethanol crude extract exhibiting the highest specific activity at 5641.25 AU / mg.

[0109] Table 6. Effects of different organic reagents on the yield of antibacterial active substances

[0110]

[0111] In summary, the optimal crude extraction method for Bifidobacterium longum B2-01 bacteriocin is the equal-volume extraction method using ethanol reagent, which exhibits the best antibacterial activity and highest specific activity. Specifically, a certain amount of fermentation supernatant obtained under the optimal culture conditions of Example 4 (pH = 6.00 ± 0.02) is freeze-dried, and then extracted with an equal volume of anhydrous ethanol using a magnetic stirrer at 600 rpm for 6 hours. After filtration, the filtrate is rotary evaporated at 45°C until completely dry to obtain the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin. Furthermore, this crude extraction method does not introduce new impurity salts into the crude extract, facilitating further separation of the antibacterial active substances.

[0112] The determination of the antibacterial activity is the same as in Example 2; the extraction rate is calculated as shown in Formula (1); the partition coefficient is calculated as shown in Formula (2); the specific activity is calculated as shown in Formula (3):

[0113]

[0114] In the formula: V1 is the volume of the organic phase, V2 is the volume of the aqueous phase, R1 is the relative potency of the organic phase, and R2 is the relative potency of the aqueous phase.

[0115]

[0116]

[0117] The method for determining the relative antibacterial potency involves using Nisin (1100 IU / mg) as a standard, dissolving it in 0.02 mol / L HCl to prepare a Nisin standard solution with a potency of 22000 IU / mL. The Nisin solution is then diluted twofold from 22000 IU / mL to 3437.5 IU / mL. Using *S. aureus* RN 4220 as an indicator bacterium, a potency standard curve (Figure 5(a)) is plotted with the net diameter of the inhibition zone as the ordinate and the logarithm of the potency value as the abscissa. The relative potency is calculated based on this standard curve. The protein concentration is determined using the BCA protein concentration assay kit. The net diameter of the inhibition zone is the diameter of the inhibition zone minus the pore size of the punch (9 mm).

[0118] Example 8: Study on the characteristics of crude bacteriocin extract from Bifidobacterium longum subsp. infantis B2-01

[0119] This embodiment uses the antibacterial activity against E. coli ATCC 25922 as an indicator to investigate the effects of six factors—UV irradiation, temperature, salt concentration, surfactant, metal ions, and pH—on the antibacterial activity of crude bacteriocin extract from *Bifidobacterium longum* subsp. *infantitidis* B2-01. The crude extract was obtained by reconstitution of the dried product obtained under the optimal crude extraction method in Example 5 with 0.2 times its original volume of distilled water. The determination of antibacterial activity was the same as in Example 2.

[0120] In the UV stability study, a certain amount of crude extract was placed under a UV lamp for 15 min, 30 min, 60 min, 120 min, and 240 min, respectively, and the antibacterial activity was measured using the untreated group as a blank control. The results are shown in Figure 6(a). Under different UV irradiation durations, only the crude extract in the 240 min irradiation group showed significantly lower antibacterial activity than the blank group, but still retained 81.42% of its antibacterial activity. The antibacterial activity of the crude extract in the other treatment groups was not significantly different from that in the blank group. This suggests that the crude extract of Bifidobacterium longum B2-01 bacteriocinolone has the potential to synergistically enhance food preservation with UV irradiation up to 120 min.

[0121] In the thermal stability study, a certain amount of crude extract was heated in a water bath at 40℃, 60℃, 80℃, and 100℃ for 30 min, and then treated under high temperature and high pressure at 121℃ for 30 min. The untreated group was used as a blank control to determine the antibacterial activity. The results are shown in Figure 6(b). When the treatment temperature was below 100℃, there was no significant difference in the antibacterial activity of the crude extract among the different temperature gradient treatment groups, and there was also no significant difference compared with the blank group. This indicates that the crude extract of Bifidobacterium longum B2-01 bacteriocinolone has good stability at temperatures below 100℃. When the treatment temperature was 121℃, the antibacterial activity of the crude extract was significantly lower than that of the blank group. This may be because high temperature and high pressure will affect the structure and function of the effective antibacterial substances in the crude extract to a certain extent, leading to a decrease in its antibacterial activity. However, the antibacterial activity after the decrease is still at a high level. Compared with the blank group, the antibacterial activity was retained by 79.34%, indicating that the crude extract of Bifidobacterium longum B2-01 bacteriocinolone has good heat resistance and has the potential to be applied to foods that require high temperature treatment.

[0122] In the salt stability study, a certain amount of crude extract was added to NaCl, and its mass fraction was adjusted to 6%, 7%, 8%, 9%, and 10%, respectively. After treatment at 37℃ for 4 hours, the untreated group was used as a blank control to determine the antibacterial activity. The results are shown in Figure 6(c). After treatment with salt concentrations of 6% to 10%, the antibacterial activity of the crude extract was significantly lower than that of the blank group, indicating that higher salt concentrations can affect the antibacterial effect of the effective antibacterial substances in the crude extract to some extent. However, although the antibacterial activity of the crude extract after treatment with each salt concentration decreased compared with the blank group, it still retained 74.18%, 74.72%, 74.72%, 77.45%, and 78.26% of the antibacterial activity, respectively. Moreover, there was no significant difference in the antibacterial activity of the crude extract treated with each concentration gradient, indicating that the crude extract of Bifidobacterium longum B2-01 bacteriocinolone has a certain possibility of being applied to high-salt foods.

[0123] In the study of surfactant stability, a certain amount of crude extract was added to Tween-20, Tween-40, Tween-80, EDTA-2Na, SDBS, and Triton X-100, respectively, and the volume fraction was adjusted to 10%. After treatment at 37℃ for 4 hours, the antibacterial activity was measured using the untreated group as a blank control. As shown in Figure 6(d), the antibacterial activity of the crude extracts treated with Tween-20, Tween-40, Tween-80, and Triton X-100 was not significantly different from that of the blank group, indicating that the crude extract of Bifidobacterium longum B2-01 bacteriocin had strong stability in the above surfactants. The antibacterial activity of the crude extract decreased significantly after SDBS treatment, indicating that SDBS seriously affects the antibacterial effect of the crude extract of Bifidobacterium longum B2-01 bacteriocin and has poor stability in SDBS. The antibacterial effect of the crude extract treated with EDTA-2Na was significantly higher than that of the blank group. It is speculated that this may be because EDTA-2Na itself has a certain antibacterial effect, and the crude extract has a synergistic effect with it after treatment, resulting in enhanced antibacterial activity.

[0124] In the study of metal ion stability, a certain amount of crude extract was added to FeSO4, MgSO4, ZnSO4, CuSO4, CaCl2, BaCl2, and MnSO4, respectively, to adjust the metal ion concentration to 5 mmol / L. After treatment at 37℃ for 4 h, the untreated group was used as a blank control to determine the antibacterial activity. The results are shown in Figure 6(e). After treatment with BaCl2, the antibacterial activity was significantly reduced. 2+ Zn 2+ Mg 2+ Mn 2+ Ca 2+ Fe 2+ The antibacterial activity of the treated crude extract was not significantly different from that of the blank group, indicating that the crude extract of Bifidobacterium longum B2-01 bacteriocinolone has strong stability in the above-mentioned metal ions; after K + The antibacterial activity of the crude extract decreased significantly after treatment, indicating that K + It affects the antibacterial effect of the crude bacteriocin extract of Bifidobacterium longum B2-01, but its antibacterial activity is still retained at 90.4%; while Cu 2+ The antibacterial effect of the treated crude extract was significantly higher than that of the control group, which is presumably due to Cu 2+ It inherently possesses a certain antibacterial effect, and the crude extract, after being processed, exhibits a synergistic effect with it, leading to enhanced antibacterial activity. Colorants, widely used in food production, play a crucial role in the color and appearance of food. Most colorants often contain a certain amount of metal ions. The crude extract of Bifidobacterium longum B2-01 bacteriocin exhibits good stability in the presence of metal ions, thus possessing the potential to be used in conjunction with colorants in food applications.

[0125] In the pH stability study, a certain amount of crude extract was taken and its pH value was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. After treatment at 37℃ for 4 hours, the pH was adjusted back to the original value, and the untreated group was used as a blank control to determine the antibacterial activity. The results are shown in Figure 6(f). When the pH value is between 5 and 7, the antibacterial activity of the crude extract is not significantly different from that of the blank group, indicating that the crude extract of Bifidobacterium longum B2-01 bacteriocin has strong stability in a slightly acidic environment. When pH < 5 or pH > 7, the antibacterial activity of the crude extract is reduced to a certain extent, but it still maintains a high level of antibacterial activity, indicating that the crude extract of bacteriocin of the present invention has good antibacterial effect in both highly acidic and alkaline environments and has good pH stability.

[0126] Example 9: Evaluation of the antibacterial activity of crude bacteriocin extract from Bifidobacterium longum subsp. infantis B2-01

[0127] This embodiment uses the agar diffusion method to investigate the antibacterial activity of crude extract of *Bifidobacterium longum* subsp. infantis B2-01 bacteriocin against *Listeria monocytogenes*, *Staphylococcus aureus*, *Enterococcus faecalis*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Lactobacillus plantarum*, *Escherichia coli*, *Salmonella enteritidis*, *Klebsiella pneumoniae*, *Cronobacter sakazakii*, *Salmonella typhimurium*, *Pseudomonas aeruginosa*, *Shewanella putrefactiveis*, *Pseudomonas fluorescens*, *Burkholderia gladioli*, *Penicillium*, *Rhizopus*, *Aspergillus flavus*, *Monascus purpureus*, *Candida*, and *Saccharomyces cerevisiae*. Each indicator bacterium was activated twice under optimal culture conditions, and the bacterial concentration of each indicator bacterium was adjusted to 10-1. 5 CFU / mL, yeast cell and mold spore concentrations were 10. 5 The antibacterial activity was determined after CFU / mL was collected, using the same method as in Example 2. The crude extract was the same as in Example 6.

[0128] The results, as shown in Table 7, indicate that the crude bacteriocin extract of *Bifidobacterium longum* B2-01 exhibits strong antibacterial effects against Gram-negative bacteria such as *Escherichia coli* and *Salmonella enteritidis*; it also shows significant antibacterial effects against Gram-positive bacteria such as *Staphylococcus aureus* and *Listeria monocytogenes*, with a slightly weaker antibacterial effect against *Enterococcus faecalis*; it has a certain degree of inhibitory effect on molds and yeasts; however, it has no antibacterial effect against *Lactobacillus rhamnosus* LR-ZB1107-01, *Lactobacillus plantarum* DMDL 9010, and *Bifidobacterium longum* B2-01 itself. Most existing studies suggest that the antibacterial substances produced by lactic acid bacteria have a relatively narrow spectrum of activity, mainly acting on bacteria closely related to the producing bacteria. However, the experimental results show that the crude bacteriocin extract produced by *Bifidobacterium longum* B2-01 has good antibacterial effects against Gram-negative bacteria and most Gram-positive bacteria, indicating its broad application prospects as a natural, novel, bio-based food preservative.

[0129] Table 7 Antibacterial spectrum of crude bacteriocin extract from Bifidobacterium longum B2-01

[0130]

[0131] Example 10: Application of crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin in the preservation of fresh tremella.

[0132] This embodiment first uses Burkholderia gladioli, a common spoilage and pathogenic bacterium found in fresh Tremella fuciformis, as the target to investigate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin. Then, the actual preservation effect of the crude extract is verified in a fresh Tremella fuciformis food system. The crude extract is the same as in Example 6.

[0133] (1) Inhibitory effect of crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin on Burkholderia gladioli CICC10574.

[0134] In the MIC assay, the crude extract was serially diluted twofold in a 96-well plate from an initial specific activity of 28206.24 AU / mg to 27.55 AU / mg. Indicator bacterial suspension was added to each well at a 1:1 ratio, with wells containing an equal volume of culture medium serving as blank controls. After thorough mixing, the plates were incubated at 37°C for 18 hours before measuring the OD. 600 No indicator bacteria growth was observed in the wells, i.e., OD. 600 The pore concentration that shows no significant change compared to the blank well is the MIC of the crude extract against B. gladioli CICC10574. The activation and culture method of the indicator bacteria solution is the same as in Example 7.

[0135] In the MBC assay, wells without indicator bacterial growth were selected. The solution in the wells was spread on LB agar plates and incubated at 37°C for 18 hours. Colony growth was observed, and the concentration of wells without colony growth was the MBC of the crude extract against B. gladioli CICC 10574.

[0136] The results are as follows Figure 7As shown, the growth of *B. gladioli* CICC 10574 was negatively correlated with the specific activity of the crude extract. When the specific activity of the crude extract was greater than 1762.89 AU / mg, no indicator bacteria were observed to grow in the treated wells, indicating that the growth of *B. gladioli* CICC 10574 was inhibited. Therefore, the MIC of the crude extract against *B. gladioli* CICC 10574 was 1762.89 AU / mg. After plating and culturing the solutions from the wells with a specific activity greater than 1762.89 AU / mg, no *B. gladioli* CICC 10574 colonies were found to grow in the treated wells when the specific activity was greater than 3525.78 AU / mg. Therefore, the MBC of the crude extract against *B. gladioli* CICC 10574 was 3525.78 AU / mg.

[0137] (2) Preservative effect of crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin on fresh tremella.

[0138] Fresh white fungus, weighing 130±5g per piece, was sprayed with 3g of the original crude extract and 3g of crude extract at a concentration of MBC after removing surface impurities. The sprayed solution was allowed to be naturally absorbed, and the fungus was air-dried and stored at 4℃ for 20 days. These were designated as group ③ (original crude extract treatment group) and group ② (MBC treatment group). A positive control (group ④) was prepared using 3g of a 1g / mol acetic acid solution containing 1% chitosan; the untreated group served as a blank control (group ①). Samples were taken every 4 days to measure various indicators of fresh white fungus quality changes, including weight loss, Hunter whiteness, and total bacterial count. The results are shown in Figure 8.

[0139] The weight loss rate of fresh white fungus is usually related to factors such as water evaporation, loss of contents, and microbial infection. The results are shown in Figure 8(a). The weight loss of the blank group ① was the most severe. Compared with the blank group ①, the treatment with crude extract of Bifidobacterium longum B2-01 bacteriocin and chitosan treatment can effectively slow down the increase in the weight loss rate of fresh white fungus, and the treatment group ③ with the original crude extract has the best effect in inhibiting the increase in weight loss rate.

[0140] The total bacterial count was used as an indicator to evaluate the microbial contamination of fresh tremella during storage. The results are shown in Figure 8(b). The total bacterial count of fresh tremella continuously increased during storage, and the total bacterial count was positively correlated with storage time. Compared to the control group ①, the rate of change in the total bacterial count of each treatment group was significantly lower than that of the control group ① (p < 0.05). The original crude extract treatment showed the strongest inhibitory effect on microorganisms during the storage of fresh tremella.

[0141] Hunter whiteness is an important parameter reflecting the color of fresh white fungus. The results are shown in Figure 8(c). The Hunter whiteness of fresh white fungus is negatively correlated with storage time. The Hunter whiteness of the blank group ① decreased the most, while the Hunter whiteness of each treatment group remained above 63. On day 20, there was no significant difference between the MBC treatment group ② and the chitosan treatment group ④, and both were lower than those of the original crude extract treatment group ③. This indicates that the original crude extract treatment group had the strongest effect on maintaining the color of white fungus.

[0142] In summary, the crude extract treatment group demonstrated a strong ability to mitigate the increase in weight loss, total bacterial count, and Hunter's whiteness of fresh white fungus during storage. This proves the application value of the crude extract of Bifidobacterium longum B2-01 bacteriocinolone in food preservation.

[0143] The weight loss rate was determined by weighing and calculated as shown in formula (4); the total bacterial count was determined in accordance with GB 4789.2-2016; the Hunter whiteness was determined by colorimeter and calculated as shown in formula (5).

[0144]

[0145] In the formula: M0 is the initial mass of fresh white fungus, M t The quality of fresh white fungus when sampled at different times.

[0146]

[0147] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin, characterized in that, Includes the following steps: (1) Fermentation culture: Take Bifidobacterium longum subsp. infantis ( Bifidobacteriu longum subsp.infantis B2-01 was inoculated into MRS liquid medium at an inoculum rate of 2% to 5% and cultured at 30 to 41°C for 32 to 40 h to obtain fermentation broth; the preservation number of Bifidobacterium longum subsp. infantis B2-01 is GDMCC No: 61911; (2) Extraction method: The fermentation broth from step (1) was centrifuged to obtain the supernatant, and the pH was adjusted to 6.0±0.

5. After freeze-drying, anhydrous ethanol was added for organic reagent extraction. The volume ratio of the supernatant to anhydrous ethanol was 1:

1. The mixture was extracted for 4-8 h at 400-800 r / min with a magnetic stirrer. After filtration, the mixture was dried. The drying was carried out by rotary evaporation of the filtrate at 40-50℃ until it was completely dried, to obtain the crude extract of Bifidobacterium longum subsp. infantis B2-01 bacteriocin. The formulation of the MRS liquid culture medium is as follows: 2±1 parts glucose, 1±0.5 parts beef extract, 1±0.5 parts casein digest, 0.4±0.2 parts yeast extract, 0.5±0.25 parts sodium acetate, 0.2±0.1 parts triammonium citrate, 0.2±0.1 parts dipotassium hydrogen phosphate, 0.108±0.05 parts Tween-80, 0.02±0.01 parts magnesium sulfate heptahydrate, 0.005±0.002 parts manganese sulfate tetrahydrate, 0.005±0.002 parts ascorbic acid, and 0.005±0.002 parts L-cysteine ​​hydrochloride. The medium is brought to a final volume of 100 parts with distilled water, and the pH is adjusted to 6.5±0.

2.

2. The preparation method according to claim 1, characterized in that, The inoculation amount in step (1) is 3.5±0.5%, the culture temperature is 36±2℃, and the culture time is 36±4 h.

3. The preparation method according to claim 1 or 2, characterized in that, Step (2) Centrifugation conditions are 2~6℃, 6000~10000 r / min for 3~7 min.

4. The preparation method according to claim 3, characterized in that, The *Bifidobacterium longum* subsp. infantis B2-01 underwent two activation cultures before inoculation. The activation culture temperature was 37±2℃ and the culture time was 16±2 h.

Images