Screening method of microalgae high-sensitization associated bacteria and targeted prevention and control

Abstract

The invention relates to the technical field of food safety, in particular to a screening method and targeted prevention and control of microalgae high-sensitization associated bacteria. The screening method disclosed by the invention comprises the following steps: (1) extracting DNA (Deoxyribose Nucleic Acid) of microalgae, and constructing a DNA library; (2) purifying the constructed DNA library; (3) carrying out genome packaging on the Clean Data; (4) predicting and clustering the assembled genes, and constructing a non-redundant gene set; (5) performing comparison and species annotation on the non-redundant gene set, and calculating the abundance of the species; (6) screening species of which the abundance ranks the first 6; and (7) predicting the potential sensitization of the coded protein in the dominant bacteria, and selecting the dominant bacteria with the most sensitization protein as the microalgae high-sensitization associated bacteria. The obtained high-sensitization associated bacterium is brucella, and the substance for specifically removing the bacterium in a targeted manner is bacteriophage vBBruSwang.

Description

Technical Field

[0001] This invention relates to the field of food safety technology, and in particular to a method for screening and targeted control of highly allergenic microalgae-associated bacteria. Background Technology

[0002] Microalgae are a potential source of high-value components with positive health effects, such as polyunsaturated fatty acids (PUFAs), pigments, vitamins, peptides, and many other bioactive compounds. Due to their high lipid accumulation capacity, strong environmental adaptability, and feasibility for industrial cultivation, they are considered one of the most promising new food resources. Because of their health-promoting properties, the consumption of microalgae has increased significantly in recent years. However, numerous reports of food allergies have occurred among microalgae consumers, raising concerns about the safety of products containing microalgae. Existing studies have found that microalgae themselves are low-allergenic foods, and the likelihood of them directly causing food allergies is very low. Therefore, exogenous allergens introduced through the cultivation system are the most likely pathway leading to microalgae allergies.

[0003] Large-scale cultivation of microalgae still faces many key technological bottlenecks, among which biofouling is particularly significant and has become a major factor restricting its industrialization. Exogenous microorganisms (especially bacteria) can easily invade the culture system through water sources, air, or inoculation materials, leading to decreased culture stability and reduced yield of the target product. The interaction between microalgae and bacteria is complex; on the one hand, the vitamins synthesized by bacteria... Essential compounds such as plant hormones promote microalgae growth; conversely, bacteria can also inhibit microalgae growth by releasing algicidal compounds that directly lyse microalgae and compete for limited nutrients. During this process, some dominant bacteria not only compete for resources but also express various conserved eukaryotic homologous proteins, such as enolases and glucose-6-phosphate isomerases. These proteins have been proven to be potential cross-reactive allergens that can induce IgE-mediated allergic reactions in humans. These allergenic proteins are difficult to completely remove during microalgae harvesting using conventional methods, and their residues significantly increase the allergenic risk of the final product, posing a potential threat, especially to individuals with food allergies. Currently, there is limited research on the expression of allergens by concomitant bacteria in microalgae culture systems.

[0004] Traditional antimicrobial methods (such as pasteurization, high-pressure processing, irradiation, and chemical disinfectants) can effectively reduce the number of microorganisms in food, but they still have significant limitations, including high initial investment, potential damage to processing equipment due to corrosiveness, adverse effects on the nutritional value of food, and the killing of many beneficial bacteria naturally present in food.

[0005] Therefore, it is urgent to solve the problem of screening dominant allergenic bacteria in microalgae culture systems and achieving precise targeted elimination of these bacteria. Summary of the Invention

[0006] The purpose of this invention is to analyze the bacterial community dynamics in microalgae culture systems, represented by *Microcystis aeruginosa*, through metagenomic sequencing, predict the intensity of bacterial sensitization, identify dominant sensitizing bacteria, and then screen for specific lysing phages to achieve precise targeted elimination of dominant sensitizing bacteria. In particular, it relates to a method for screening and targeted control of highly sensitizing symbiotic bacteria associated with microalgae.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for screening highly allergenic microalgae-associated bacteria, comprising the following steps: (1) Extract DNA from microalgae and construct a DNA library; (2) The constructed DNA library was purified to obtain Clean Data; (3) Perform genome assembly on the Clean Data to obtain the assembled genes; (4) Predict the assembled genes and perform cluster analysis. Select the longest gene in each class as the representative sequence to construct a non-redundant gene set. At the same time, quantify the non-redundant gene set to obtain gene abundance values. (5) Compare the non-redundant gene set with the NR database, annotate the species, and calculate the abundance of the species; (6) Select the top 6 species by abundance to obtain the dominant bacterial genera; (7) Predict the potential allergenicity of the encoded proteins in the dominant bacterial genera and select the dominant bacterial genera with the largest total number of allergenic proteins as microalgae highly allergenic symbiotic bacteria.

[0008] Preferably, the purification process in step (2) includes: removing reads containing sequencing adapter sequences; removing low-quality sequences; finding overlaps in each pair of reads and appropriately correcting inconsistent bases within the interval; aligning reads to the host genome and removing reads with high alignment similarity originating from the host genome or contaminating the genome.

[0009] Preferably, the genome assembly method in step (3) is performed using MEGAHIT software.

[0010] Preferably, the prediction method in step (4) is: prediction using METAProdigal; The clustering analysis method in step (4) is as follows: CD-HIT software is used, and the parameters for clustering analysis are: 93~97% identity, 88~92% coverage; The quantitative method in step (4) is to use Salmon software.

[0011] Preferably, the comparison method in step (5) is BLASTP.

[0012] Preferably, the prediction method in step (7) is to use the AllerCatPro 2.0 tool for prediction.

[0013] This invention also provides that the highly allergenic symbiotic bacteria of microalgae obtained by the screening method described above are Brucella spp. ( Brucella anthropi ).

[0014] This invention also provides the application of bacteriophage vB_BruS_wang in antagonizing Brucella growth, characterized in that the bacteriophage vB_BruS_wang is Brucella arthropi Phage vB_BruS_wang is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252464, on November 6, 2025.

[0015] This invention also provides the application of bacteriophage vB_BruS_wang in reducing the production of highly allergenic substances in microalgae, characterized in that the bacteriophage vB_BruS_wang is Brucella arthropi Phage vB_BruS_wang is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252464, on November 6, 2025.

[0016] This invention also provides the application of bacteriophage vB_BruS_wang in reducing the allergenicity of microalgae, characterized in that the bacteriophage vB_BruS_wang is Brucella arthropi Phage vB_BruS_wang is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252464, on November 6, 2025.

[0017] The present invention has the following advantages: This invention uses metagenomic sequencing to analyze bacterial community dynamics in microalgae culture systems, represented by *Microcystis aeruginosa*, predicts bacterial sensitization intensity, and identifies *Brucella* as the dominant sensitizing bacterium. Brucela anthropi Then, specific lysing phage vB_BruS_wang is screened to achieve precise targeted elimination of dominant sensitizing bacteria.

[0018] Preservation Instructions

[0019] bacteriophage vB_BruS_wang is Brucella arthropiPhage vB_BruS_wang is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252464, on November 6, 2025. Attached Figure Description

[0020] Figure 1 A bar chart of species abundance (C, G, Y represent three different groups of samples); Figure 2 A pie chart of species abundance; Figure 3 This represents the optimal infection multiple of the bacteriophage; Figure 4 The growth trend of marine microcystis; Figure 5 The growth trends of Brucella and Paracoccus; Figure 6 This represents changes in bacteriophage titers and the growth trends of Brucella and Paracoccus. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1

[0023] 1. Screening and identification of highly allergenic microalgal symbiotic bacteria

[0024] 1.1 Metagenomic sequencing and genome assembly

[0025] (1) Sample setup. 10 mL of *Microcystis aeruginosa* algal solution in the logarithmic growth phase was collected from a six-channel photobioreactor, and three parallel samples were set up. (2) DNA extraction. DNA was extracted from 500 μL of algal solution using the EZNA™ Mag-Bind Soil DNA Kit. (3) DNA library construction. A DNA library was constructed by DNA fragmentation, end repair, adapter ligation, and PCR amplification. The library size was detected by 2% agarose gel electrophoresis, and the library concentration was determined using a Qubit4.0 fluorescence quantitative PCR instrument. (4) Data quality control. The raw sequencing data was purified to obtain Clean Data. Reads containing sequencing adapter sequences were removed; low-quality sequences were removed, including sequences with a large number of ambiguous bases and paired-end reads with a length of less than 35 nt; overlap of each pair of reads was identified, and inconsistent bases within the interval were appropriately corrected. (5) Genome assembly. The Clean Data was assembled and analyzed using MEGAHIT software. (6) Gene prediction and abundance information. Metagenomic genes were predicted de novo using METAProdigal, and clustering was performed using CD-HIT software (parameters: 95% identity, 90% coverage). The longest gene in each cluster was taken as the representative sequence to construct a non-redundant gene set. Salmon software was used for quantification, and the obtained TPM value is the standardized gene abundance value. (7) Species annotation and species abundance calculation. Using BLASTP, the non-redundant gene set was compared with the NR database (https: / / ftp.ncbi.nlm.nih.gov / blast / db / ), and species annotations were obtained through the taxonomic information database corresponding to the NR database. The sum of gene abundance corresponding to a species is the abundance of that species.

[0026] 1.2 Preliminary identification of highly allergenic commensal bacteria

[0027] Based on metagenomic sequencing and genome assembly results, the six most abundant species in the *Microcystis aeruginosa* culture system were identified as dominant genera. The AllerCatPro 2.0 tool was used to predict the potential sensitization of proteins encoded by these dominant genera. The dominant genera with the highest total number of sensitizing proteins were selected as highly sensitizing concomitant bacteria. The prediction results are shown in Table 1.

[0028] 1.3 Isolation, Identification, and Extraction of Associated Bacteria

[0029] Take marine micrococcus pluvialis in the logarithmic growth phase ( Nannochloropsis oceanicaThe algal solution, diluted with 0.01 M phosphate buffer, was evenly spread onto trypsin-soybean agar (TSA) plates. After incubating the plates at 27°C for 48 hours, single colonies with different morphological characteristics were picked for purification and culture, and the abundance of each colony type was recorded. The purified strains were then identified by 16S rDNA sequencing by Sangon Biotech (Shanghai) Co., Ltd.

[0030] 1.4 Verification of highly allergenic commensal bacteria

[0031] 1.4.1 Extraction of total protein from Brucella bacteria

[0032] Take OD 600 100 mL of Brucella bacterial culture with a pH of 1.0 was centrifuged at 8000 × g for 20 min in a low-temperature high-speed centrifuge. The bacterial pellet was collected and disrupted using a high-pressure cell disruptor at 700 bar and 5 °C for 10 min. After disruption, the pellet was centrifuged again, and the supernatant was collected to obtain the Brucella total protein extract with a concentration of 2 mg / mL.

[0033] 1.4.1 Preparation of mouse anti-brucellosis total protein serum

[0034] Female Balb / c mice were used for immunization. Immunization was performed via intraperitoneal injection and gavage challenge, and mice were divided into an experimental group and a blank control group. The blank control group was injected with sterile PBS, while the experimental group was injected with 200 μL of a homogenized solution obtained by mixing Brucella total protein with aluminum hydroxide adjuvant. Mice were intraperitoneally immunized on days 7, 14, 21, and 28, and challenged by gavage on day 35. On day 36, blood was collected from the orbital cavity of the mice, and they were then sacrificed by cervical dislocation. The mouse blood was incubated on ice for 3 hours, centrifuged at 8000×g for 10 min at 4°C, and the collected serum was stored at -80°C.

[0035] 1.4.2 Indirect ELISA Analysis

[0036] The specific antibody IgE in serum was detected by indirect enzyme-linked immunosorbent assay. (1) Coating. Brucella total protein antigen was diluted to 20 μg / mL with 0.05 mol / L pH 9.6 CBS and coated onto 96-well plates, 100 μL per well, overnight at 4℃. (2) Washing: The next day, the ELISA plate was removed and placed in a plate washer, the coating solution was discarded, and the plate was washed three times with PBST for 3 min each time, and then patted dry. (3) Blocking: The ELISA plate was filled with blocking solution and blocked at 37℃ for 2 h, washed three times, and then patted dry. (4) Primary antibody incubation: Mouse anti-brucellosis total protein IgE antibody serum was diluted 1000 times, the blank group was 0.01 mol / L pH 7.4 PBS, and the control group was negative serum, 100 μL per well, incubated at 37℃ for 1 h, washed three times with PBST, and then patted dry. (5) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgE antibody (1:10000 dilution) to each well, incubate at 37℃ for 1 h, then wash three times with PBST and pat dry. (6) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37℃ in the dark for 10 min. (7) Termination: Add 50 μL of stop solution to each well to terminate the reaction. (8) Detection: Measure the absorbance at 450 nm.

[0037] Results: Metagenomic sequencing yielded 231,973,875 raw reads, with a total data volume of 34,796.08 Mbp. After quality control, the number of raw reads and the total data volume were 224,485,690 and 33,617.84 Mbp, respectively. Subsequent assembly produced 19,532 contigs with a total length of 82,490,856 bp. A total of 2,557 species were identified from all samples. Based on the relative abundance tables at different taxonomic levels, the top 10 species by relative abundance in each sample were selected, and the remaining species were grouped into the "Other" category. Figure 1 The top 6 species are considered the dominant genera.

[0038] Table 1. Results of online prediction of potential sensitization by proteins from 6 dominant bacterial genera.

[0039] Table 1 shows Brucella bacteria. (Brucella anthropi) Brucella had the highest total number of sensitizing proteins (204,938), with 768 strongly sensitizing proteins and 6,568 weakly sensitizing proteins detected, accounting for 3.57% of the total sensitizing proteins. Comparative analysis showed that Brucella posed a significantly higher sensitizing risk than the other five species. Based on this, Brucella was identified as the target strain for removal.

[0040] Indirect ELISA analysis showed that the negative serum control group had OD 450=0.050, OD450=0.354 for the Brucella total protein experimental group. Compared with the negative serum control group, the serum IgE secretion level of mice in the Brucella total protein experimental group was significantly increased, demonstrating that the protein has sensitizing properties.

[0041] Example 2

[0042] 2. Targeted control of highly allergenic commensal bacteria

[0043] 2.1 Isolation and purification of bacteriophages

[0044] Wastewater samples were collected from Zhaike Wharf, Lianxin Fishing Port, Huangdao District, Qingdao City, Shandong Province. Samples were centrifuged at 4℃ and 6000 rpm for 20 min to remove impurities. The supernatant was poured into clean test tubes and filtered through a 0.22 μm filter membrane to remove bacteria. 5 mL of semi-solid agar was added to a sterile test tube. 100 μL of Brucella anthropi (OD600 0.5, purchased from the China Marine Microbial Culture Collection Center, accession number 1A16327) and 100 μL of filtered virus sample (filtrate from wastewater filtered through a 0.22 μm filter membrane) were mixed and transferred to a sterile test tube. The mixture was poured into TSA plates. The plates were incubated upside down at 27℃ for 24 h until plaque formation was observed. A single plaque (1 PFU) (phage) was selected, suspended in 1 mL of SM buffer, and then serially diluted 10-fold. In a sterile test tube, mix 100 μL of phage liquid with 100 μL of Brucella bacteria, pour into a TSA plate, and incubate at 27°C until visible plaques appear. Repeat the above purification process until all plaques exhibit consistent morphological characteristics, thus obtaining the phage.

[0045] 2.2 Determination of the optimal multiple of infection (MOI) for bacteriophages

[0046] The isolated phages were inoculated into 5 mL of solution at MOI gradients of 10, 1, 0.1, 0.01, 0.001, and 0.0001. In a CFU / mL Brucella culture, after static adsorption at 27°C for 30 min, the culture was transferred to a shaker and incubated at 200 rpm for 24 h. The phage titer was finally determined using the double-layer agar plate method. Results are as follows: Figure 3 As shown.

[0047] 2.3 Biocontrol of Brucella by Bacteriophages in Marine Micrococcus neoformans Systems

[0048] First, the initial concentration of Brucella in the *Microcystis aeruginosa* system was determined. Serial dilutions of the algal solution were plated on tryptone-soy agar (TSA) plates and incubated at 27°C for 24 h. Colony forming units (CFU) were then counted. Based on the optimal multiple of infection (MOI=1), a final concentration of 1.0 × 10⁻⁶ phages was added to the system. Phage suspensions of PFU / mL were prepared in triplicate for each group. Samples were taken every 24 h for the following analyses: (1) The absorbance of the algal solution was measured using a UV-Vis spectrophotometer. (2) After gradient dilution of the algal solution, it was spread on TSA plates and incubated at 27℃ for 24 h to count viable Brucella bacteria; (3) The phage titer (PFU / mL) was determined by double-layer agar method. The biocontrol effect of phage is as follows: Figures 4-6 As shown.

[0049] Results: Using Brucella as the host bacterium, bacteriophage vB_BruS_wang was isolated, with the Latin name [missing information]. Brucella arthropi Phage vB_BruS_wang is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO:M20252464, on November 6, 2025.

[0050] The MOI optimization experiment of bacteriophage vB_BruS_wang infecting Brucella showed that the highest titer (3.0 × 10⁻⁶) was obtained when MOI=1. PFU / mL).

[0051] Under both phage-added and phage-free conditions, *Microcystis aeruginosa* exhibited similar growth trends (P>0.05). This result indicates that the introduction of phages did not significantly affect the growth of *Microcystis aeruginosa*.

[0052] After the addition of bacteriophage, a strong bactericidal effect against Brucella was observed. The presence of bacteriophage led to a rapid and continuous decrease in the viable Brucella count, from an initial 6.25 log CFU / mL to 1.26 log CFU / mL at the end of the culture, indicating a low level of contamination. Conversely, in the control group without bacteriophage, the Brucella concentration increased slightly, eventually reaching 6.88 log CFU / mL, which was 5.62 log CFU / mL higher than the bacteriophage-treated group. The growth dynamics of Paraacoccus sediminilitoris, the second dominant bacterium in the marine microalgae culture, were analyzed. The results showed that Paraacoccus sediminilitoris did not overproliferate due to the decrease in Brucella count, and its growth was not significantly different from the control group (P>0.05).

[0053] Throughout the sterilization process, the phage titer initially increased and then slowly decreased, eventually dropping to 7.03 log PFU / mL. Figure 6 Although the phage titer remained relatively stable throughout the treatment, complete eradication of Brucella was not achieved. These results indicate that the efficiency of phage in successfully infecting and lysing host bacteria is highly dependent on the phage-to-bacteria ratio.

[0054] As can be seen from the above embodiments, the present invention analyzes the bacterial community dynamics in a microalgae culture system represented by marine microalgae micrococcus through metagenomic sequencing, predicts the intensity of bacterial sensitization, identifies Brucella as the dominant sensitizing bacterium, and then screens for the specific lysing bacteriophage vB_BruS_wang to achieve precise targeted elimination of the dominant sensitizing bacterium.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for screening of high-sensitization associated bacteria of microalgae, characterized by, It comprises the following steps: (1) Extracting DNA of microalgae, constructing DNA library; (2) Purifying the constructed DNA library to obtain Clean Data; (3) Assembling the Clean Data to obtain assembled genes; (4) Predicting the assembled genes, performing clustering analysis, selecting the longest gene in each class as a representative sequence, and constructing a non-redundant gene set; meanwhile, quantifying the non-redundant gene set to obtain gene abundance values; (5) Aligning the non-redundant gene set with the NR database, annotating the species, and calculating the abundance of the species; (6) Screening the top 6 species in the abundance of the species to obtain dominant bacterial genera; (7) Predicting the potential allergenicity of the encoded proteins in the dominant bacterial genera, and selecting the dominant bacterial genus with the most allergenic proteins as the microalgae highly allergenic symbiotic bacteria.

2. The screening method according to claim 1, characterized in that, The purification method of step (2) comprises: rejecting reads containing sequencing adapter sequences; rejecting low-quality sequences; finding the overlap of each pair of reads and appropriately correcting the inconsistent bases in the interval; aligning the reads to the host genome to remove reads derived from the host genome or pollution with high similarity.

3. The screening method according to claim 1, characterized by, The method of step (3) for gene assembly is to use MEGAHIT software.

4. The screening method according to claim 1, characterized by, The method of step (4) for prediction is to use METAProdigal for prediction. The method of step (4) for clustering analysis is to use CD-HIT software, and the parameters for clustering analysis are: 93-97% identity, 88-92% coverage. The method of step (4) for quantification is to use Salmon software.

5. The screening method according to claim 1, wherein The method of step (5) for alignment is BLASTP.

6. The screening method according to claim 1, wherein The method of step (7) for prediction is to use AllerCatPro 2.0 tool for prediction.

7. The microalgae with high allergenicity obtained by the screening method according to any one of claims 1 to 6 are Brucella spp. ( Brucella anthropi ).

8. Use of the bacteriophage vB_BruS_wang in antagonizing the growth of Brucella, characterized in that, The phage vB_BruS_wang is Brucella arthropi phage vB_BruS_wang, deposited with the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on November 06, 2025, and assigned accession number CCTCC NO: M20252464.

9. Use of the bacteriophage vB_BruS_wang in reducing the production of highly allergenic substances in microalgae, characterized in that, The phage vB_BruS_wang is Brucella arthropi phage vB_BruS_wang, deposited with the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on November 06, 2025, and assigned accession number CCTCC NO: M20252464.

10. Use of the bacteriophage vB_BruS_wang in reducing the allergenicity of microalgae, characterized in that, The phage vB_BruS_wang is Brucella arthropi phage vB_BruS_wang, deposited with the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on November 06, 2025, and assigned accession number CCTCC NO: M20252464.

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