A bacteriophage of lactobacillus plantarum and application thereof in fermented vegetables

Abstract

The application provides a plant lactobacillus phage and application thereof in fermented vegetables, and belongs to the technical field of microbial fermentation. The plant lactobacillus phage SAASZN03045 has a preservation number GDMCC 67684-B1. The plant lactobacillus phage SAASZN03045 provided by the application has high specificity, only plays a lysis role on specific plant lactobacillus strains, does not damage other microorganisms in the fermented vegetables, is beneficial to maintaining the balance of the fermentation bacterial population, has the effects of promoting the rapid production of lactic acid by the bacterial population in the fermented vegetables, reducing the acetic acid content and improving the sour and fragrant flavor, is suitable for the production environment of the fermented vegetables, can solve the problems of long fermentation period, slow acid production and insufficient aroma in the fermented vegetable industry, and has wide application scenarios and market development potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and in particular relates to a plant lactobacillus phage and its application in fermented vegetables. Background Technology

[0002] Fermented vegetables are a representative traditional fermented food. With their crisp and palatable texture and rich, tangy flavor, they are not only a popular side dish but also a distinctive condiment used in cooking, possessing both nutritional and flavoring value and holding an important position in the food industry. Currently, as the food industry strives for standardization, large-scale production, and high quality, the inherent drawbacks of traditional natural fermentation are becoming increasingly apparent, posing a core bottleneck to the standardized and industrialized development of the fermented vegetable industry: natural fermentation cycles are lengthy, and the fermentation process is difficult to control precisely; the open fermentation system is prone to the growth of unwanted bacteria, posing not only food safety risks but also interfering with normal fermentation metabolism; simultaneously, there are problems such as slow acid production rates, insufficient accumulation of flavor substances, and a thin product flavor, failing to meet the stringent requirements of modern industrial production for product quality uniformity and stability.

[0003] The fermentation process of fermented vegetables relies on a multi-species synergistic fermentation system of lactic acid bacteria and yeast. Lactic acid bacteria are the core functional microbial community that dominates the fermentation process, with *Lactobacillus* species being the core organism within the system. Lactic acid bacteria, represented by *Lactobacillus plantarum*, can efficiently metabolize carbohydrates in vegetable raw materials, converting them into organic acids such as lactic acid and acetic acid. This not only imparts a refreshing, tangy flavor to the fermented vegetables but also lowers the pH of the system, inhibiting the growth of unwanted microorganisms and ensuring the safety and stability of the fermentation process. Currently, modern fermented vegetable production commonly employs a directional fermentation process inoculated with *Lactobacillus plantarum*, aiming to replace traditional natural fermentation and achieve controllable fermentation processes while improving product quality.

[0004] However, in actual production, the microecological environment within fermented vegetable systems is complex. The coexistence and competition of multiple microbial species, along with the continuous dynamic changes in physicochemical factors such as temperature, pH, and nutrient components during fermentation, easily lead to fluctuations in the number of viable *Lactobacillus plantarum* bacteria and a decline in fermentation performance. This results in problems such as unstable acid production, uneven flavor, and uncontrolled fermentation cycles, ultimately causing inconsistent quality in the finished fermented vegetables and making standardized mass production difficult. Therefore, developing specific technologies to regulate the growth, reproduction, metabolic acid production, and fermentation performance of *Lactobacillus plantarum* within fermented vegetable systems, and solving the industry problem of unstable fermentation quality, is of crucial practical significance for promoting the upgrading of the fermented vegetable industry and achieving standardized industrial production.

[0005] Current conventional control techniques for *Lactobacillus plantarum* primarily target physicochemical parameters such as temperature, pH, ionic strength, and salinity. However, these methods lack specificity. Because the response mechanisms of various lactic acid bacteria within a fermentation system to environmental stress are highly similar, traditional control methods, while acting on *Lactobacillus plantarum*, indiscriminately affect the growth and metabolism of other lactic acid bacteria in the system, disrupting the original microbial balance and exacerbating the instability of the fermentation system. This makes precise and efficient targeted control impossible.

[0006] To address the common pain points in current fermented vegetable production, such as slow fermentation, insufficient acid production, and poor flavor caused by the unstable growth and fermentation performance of *Lactobacillus plantarum*, a technology for targeted regulation of acid production in fermented vegetables based on *Lactobacillus plantarum* bacteriophages has been developed. This technology not only effectively compensates for the shortcomings of existing regulation technologies but also aligns with the trend of green, safe, and efficient fermented food production, possessing broad prospects for industrial application and market value. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a plant lactobacillus phage and its application in fermented vegetables.

[0008] This invention provides a plant lactobacillus phage SAASZN03045, the preservation number of which is GDMCC 67684-B1.

[0009] Preferably, the titer of the *Lactobacillus plantarum* bacteriophage SAASZN03045 is 10. 8 ~10 9 PFU / mL.

[0010] Preferably, the physicochemical stability range of the *Lactobacillus plantarum* phage SAASZN03045 is as follows: temperature 0℃~40℃, pH value 2~12, NaCl concentration 0%~18%.

[0011] This invention provides the application of the aforementioned plant lactobacillus phage SAASZN03045 in regulating plant lactic acid bacteria fermentation of vegetables.

[0012] This invention provides a method for preparing fermented vegetables, comprising the following steps: After inoculating the vegetable brine mixture with a compound fermentation agent, the plant lactobacillus phage SAASZN03045 was inoculated, and fermentation was carried out at 20-25℃ for 2-5 days to obtain fermented vegetables.

[0013] Preferably, the compound fermentation agent comprises strains in the following mass ratio: *Lactobacillus plantarum* SAASZN01137: *Lactobacillus namurti* SAASZN01047: *Lactobacillus bruneri* SAAS-B-MRS-20200906-11: *Lactobacillus brevis* SAASZN00575: *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 in a ratio of (1~1.2): (0.8~1): (0.9~1.1): (1~1.2):(0.1~1); the viable cell concentrations of *Lactobacillus plantarum* SAASZN01137, *Lactobacillus namurti* SAASZN01047, *Lactobacillus bruneri* SAAS-B-MRS-20200906-11, *Lactobacillus brevis* SAASZN00575, or *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 are each 10... 6 ~10 8 CFU / mL.

[0014] Preferably, the inoculum amount of the compound fermentation agent is 2%~5% (w / w).

[0015] Preferably, the titer of the *Lactobacillus plantarum* bacteriophage SAASZN03045 is 10. 8 ~10 9 PFU / mL.

[0016] Preferably, the ratio of vegetables to brine in the vegetable-salt mixture is 1g:(1~3)mL, and the salt content of the brine is 1%~8%.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a plant lactobacillus phage, SAASZN03045, which is highly specific and exerts a lytic effect only on specific plant lactobacillus strains without damaging other microorganisms in fermented vegetables. This helps maintain the balance of the fermentation community and promotes the rapid production of lactic acid, reduces acetic acid content, and enhances the sour and aromatic flavor of fermented vegetables. This phage can maintain high activity within a temperature range of 0℃~40℃, pH value of 2~12, and NaCl concentration of 0%~18%, making it suitable for the fermentation environment of vegetables. It can solve the industry problems of long fermentation cycle, slow acid production, and insufficient aroma in fermented vegetables, and has a wide range of application scenarios and market development potential. Attached Figure Description

[0018] Figure 1 This is a phage plaque image of the plant lactobacillus phage SAASZN03045 of the present invention; Figure 2 This is a transmission electron microscope image of the plant lactobacillus phage SAASZN03045 of this invention; Figure 3This is a complete genome loop of the plant lactobacillus phage SAASZN03045 of this invention; Figure 4 This is the optimal infection multiple diagram for the *Lactobacillus plantarum* bacteriophage SAASZN03045 of the present invention; Figure 5 This is a one-step growth curve of the plant lactobacillus phage SAASZN03045 of the present invention. Figure 6 Temperature tolerance diagram of the plant lactobacillus phage SAASZN03045 of this invention; Figure 7 This is a pH tolerance diagram of the plant lactobacillus phage SAASZN03045 of the present invention; Figure 8 This is a NaCl tolerance diagram of the plant lactobacillus phage SAASZN03045 of this invention; Figure 9 The reducing sugar content of radish kimchi fermented with bacteriophage + compound lactic acid bacteria, fermented with Lactobacillus plantarum, fermented with compound lactic acid bacteria, and fermented with (-) Lactobacillus plantarum and compound lactic acid bacteria was determined. Figure 10 The total acid content of fermented radish kimchi was determined by the following methods: bacteriophage + compound lactic acid bacteria group, plant lactobacillus group, compound lactic acid bacteria group, and (-) plant lactobacillus compound lactic acid bacteria fermented radish kimchi. Figure 11 pH values ​​for fermented radish kimchi using bacteriophage + compound lactic acid bacteria, fermented radish kimchi using Lactobacillus plantarum, fermented radish kimchi using compound lactic acid bacteria, and fermented radish kimchi using (-) Lactobacillus plantarum and compound lactic acid bacteria; Figure 12 Lactic acid bacteria counts were performed on radish kimchi fermented with bacteriophage + compound lactic acid bacteria, fermented with Lactobacillus plantarum, fermented with compound lactic acid bacteria, and fermented with (-) Lactobacillus plantarum and compound lactic acid bacteria. Figure 13 Organic acid composition diagrams for radish kimchi fermented with bacteriophage + compound lactic acid bacteria, fermented with Lactobacillus plantarum, fermented with compound lactic acid bacteria, and fermented with (-) Lactobacillus plantarum and compound lactic acid bacteria. Figure 14 The volatile flavor components of radish kimchi fermented with bacteriophage + compound lactic acid bacteria, fermented with Lactobacillus plantarum, fermented with compound lactic acid bacteria, and fermented with (-) Lactobacillus plantarum and compound lactic acid bacteria are shown in the figure.

[0019] Biological Preservation Instructions

[0020] Lactobacillus plantarum phage ( Lactiplantibacillus plantarumphage)SAASZN03045 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 15, 2026. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The accession number is GDMCC NO: 67684-B1. Lactobacillus plantarum ( Lactiplantibacillus plantarum SAASZN01137 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 15, 2026. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The accession number is GDMCC NO: 67681. Lactobacillus namuri ( Levilactobacillus namurensis SAASZN01047 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 15, 2026. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The accession number is GDMCC NO: 67682. Lactobacillus bruneri ( Lentilactobacillus buchneri SAAS-B-MRS-20200906-11 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 19, 2023. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The accession number is GDMCC NO: 63484. Lactobacillus brevis ( Levilactobacillus brevis SAASZN00575 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on January 15, 2026. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou. The accession number is GDMCC NO: 67683. Ethanol-resistant Pediococcus ( Pediococcus ethanolidurans SAAS-B-MRS-20201030-3 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 19, 2023. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, and the accession number is GDMCC NO. 63487. Detailed Implementation

[0021] This invention provides a plant lactobacillus phage SAASZN03045, the preservation number of which is GDMCC 67684-B1.

[0022] In this invention, the titer of the *Lactobacillus plantarum* bacteriophage SAASZN03045 is 10. 8 ~10 9PFU / mL; the physicochemical stability range of the *Lactobacillus plantarum* phage SAASZN03045 is as follows: temperature 0℃~40℃, pH value 2~12, NaCl concentration 0%~18%. In this invention, the *Lactobacillus plantarum* phage SAASZN03045 is isolated from fermented vegetables.

[0023] This invention provides the application of the plant lactobacillus phage SAASZN03045 in regulating plant lactic acid bacteria fermentation of vegetables, especially in regulating the acid production process of fermented vegetables.

[0024] The present invention also provides a method for preparing fermented vegetables, comprising the following steps: inoculating a compound fermentation agent into a vegetable brine mixture, then inoculating the plant lactobacillus phage SAASZN03045, and fermenting at 20~25℃ for 2~5 days to obtain fermented vegetables.

[0025] In this invention, the compound fermentation agent comprises the following strains in the following mass ratio: *Lactobacillus plantarum* SAASZN01137: *Lactobacillus namurti* SAASZN01047: *Lactobacillus brunelli* SAAS-B-MRS-20200906-11: *Lactobacillus brevis* SAASZN00575: *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 (1~1.2): (0.8~1): (0.9~1.1): (1~1.2): (0.1~1), preferably 1:1:1:1:1; the viable bacterial concentrations of *Lactobacillus plantarum* SAASZN01137, *Lactobacillus namurti* SAASZN01047, *Lactobacillus brunelli* SAAS-B-MRS-20200906-11, *Lactobacillus brevis* SAASZN00575, or *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 are preferably 10. 6 ~10 8 CFU / mL, more preferably 5×10⁻⁶ 6 ~5×10 7 CFU / mL.

[0026] In this invention, the plant lactobacillus ( Lactiplantibacillus plantarum SAASZN01137 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 67681; the Lactobacillus namurti mentioned therein ( Levilactobacillus namurensis SAASZN01047 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 67682; the Lactobacillus brunelli (…) mentioned herein… Lentilactobacillus buchneri SAAS-B-MRS-20200906-11 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 63484; the Lactobacillus brevis mentioned therein ( Levilactobacillus brevis SAASZN00575 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC 67683; the ethanol-resistant Pediococcus described therein ( Pediococcus ethanolidurans SAAS-B-MRS-20201030-3 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.63487.

[0027] In this invention, the inoculum amount of the compound fermentation agent is preferably 2%~5% (w / w), more preferably 3%~4% (w / w); the ratio of vegetables to brine in the vegetable-salt mixture is preferably 1g:(1~3)mL, more preferably 1g:(1.5~2.5)mL; the salt content of the brine is 1%~8%, preferably 2%~7%. In this invention, the titer of the *Lactobacillus plantarum* phage SAASZN03045 is preferably 10. 8 ~10 9 PFU / mL. This invention does not limit the type of vegetable used, but cruciferous vegetables are preferred.

[0028] 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.

[0029] Example 1

[0030] Isolation, purification and identification of Bacteroides plantarum phage SAASZN03045

[0031] 1) Activation of host bacteria

[0032] Take the host bacterium *Lactobacillus plantarum* SAASZN01137 stored at -80℃, streak it on an MRS solid plate, and incubate it statically at 37℃ for 24 hours. Pick a single colony with uniform morphology from the plate, transfer it to MRS liquid medium, and incubate it at 37℃ for 24 hours. Then, inoculate 5 mL of fresh MRS liquid medium at a 2% (v / v) inoculation ratio, and incubate it at 37℃ for 16 hours. After the incubation, store it in a 4℃ refrigerator for later use.

[0033] 2) Sample collection and pretreatment

[0034] Take an appropriate amount of fermented vegetable brine and centrifuge it at 5000 rpm for 10 min. Collect the supernatant and filter it through a 0.22 μm sterile microporous membrane to obtain a sterile fermentation broth sample for later use.

[0035] 3) Phage enrichment

[0036] Take 4 mL of the treated fermentation broth sample and mix it thoroughly with an equal volume of MRS-Ca liquid medium. Add the activated host bacterial solution at a ratio of 1:40 (v / v). At the same time, set up a blank control group with an equal volume of sterile physiological saline instead of the fermentation broth sample. Incubate the mixture at 37℃ for 12-16 h and observe the changes in turbidity. Finally, collect the clear phage enrichment solution.

[0037] 4) Preparation of phage plaques

[0038] The clarified phage enrichment solution was serially diluted 10-fold with MRS liquid medium. Then, 4 mL of MRS-Ca semi-solid medium (45℃–50℃) was thoroughly mixed with 100 μL of the serially diluted phage solution and 100 μL of the activated host bacterial solution, respectively. This mixture was then quickly poured onto the surface of the MRS solid medium. After the semi-solid medium had completely cooled and solidified, the plates were incubated at 37℃ for 24 h. After incubation, the growth of phage plaques on the plates was observed. The plaque results are shown below. Figure 1 As shown, *Lactobacillus plantarum* phage SAASZN03045 forms circular, clear phage plaques on a double-layer agar plate. Figure 1 The diameter is 1~2 mm, and the potency is 8.331g (PFU / mL).

[0039] 5) Phage purification

[0040] Using a sterile pipette tip, precisely pick up a single, clearly morphologically distinct phage plaque from the plate and rinse it thoroughly in 1 mL of MRS-Ca liquid culture medium to completely elute the phage into the culture medium. Then, centrifuge at 5000 rpm, collect the supernatant, and filter it through a 0.22 μm sterile microporous membrane for sterilization. Finally, take 4 mL of MRS-Ca liquid culture medium, add 100 μL of the filtered phage supernatant and 100 μL of host bacterial culture, and incubate at 37°C for 24 h. After incubation, centrifuge and filter the culture medium again, and use the filtrate to prepare bilayer plates. Repeat the above steps 5 times until uniformly sized and morphologically consistent phage plaques grow on the plate, thus obtaining the purified single phage.

[0041] 6) Electron microscopic observation of bacteriophages

[0042] 20 μL of purified phage solution was dropped onto an electron microscope grid and allowed to stand at room temperature for 10 min to adsorb. Excess droplets were gently blotted away with filter paper. After slightly drying, 20 μL of uranium acetate staining solution was dropped onto the grid and allowed to stain for 5 min. After staining, residual staining solution was removed, and the grid was dried under an incandescent lamp. Finally, the morphology of the phage was observed and images were acquired using a transmission electron microscope (JEM1400). The morphology of the phage observed under the transmission electron microscope is shown below. Figure 2As shown, the *Lactobacillus plantarum* phage SAASZN03045 consists of a head and a tail, with a total length of approximately 208 nm. The head is roughly rectangular in shape, with a diameter of approximately 65 nm; a non-retractable long tail was observed in the tail, with a length of approximately 143 nm. Based on morphology, this phage is identified as belonging to *Siphoviridae* genus *Caudovirales*.

[0043] 7) Whole genome sequencing and analysis of Lactobacillus bacteriophages

[0044] Phage samples were sent to a biotechnology company for whole-genome sequencing. Concentrated phage samples were filtered through a 0.22 μm filter membrane. The filtrate was treated with 200 U Benzo DNase (NOVOPROTEIN SCINETIFIC INC) and 0.1 mg / ml RNase A (Sangon Biotech), followed by heat inactivation of the DNase at 65°C for 10 min. Phage genomic DNA was extracted using the Qiagen MinElute Virus Spin Kit according to the manufacturer's instructions. DNA concentration was detected using the Equalbit1x dsDNA HS Assay Kit (Vazyme Biotech Co., Ltd) on a Qubit 3.0 fluorometer (ThermoFisher Scientific), and genomic DNA size was determined by 0.8% agarose gel electrophoresis. Libraries were constructed using the VAHTS® Universal Plus DNA Library Preparation Kit (Vazyme Biotech Co., Ltd.), and the libraries were quality checked using an Agilent 4200 Bioanalyzer. After passing the quality check, PE150 sequencing was performed on the DNBSEQ-T7 platform.

[0045] The raw sequencing data obtained from sequencing were filtered and quality-controlled using FASTP to obtain clean reads. The clean reads were then assembled using MetaSPAdes software for de novo assembly. Different k-mer lengths were selected for testing to obtain the optimal assembly results. The clean reads were then aligned to the assembled genome sequence for statistical coverage using BWA software. The genome was annotated with coding genes and tRNAs using Prokka. The protein sequences were then aligned with the NR library using BLASTP to obtain the sequence information with high similarity for each gene in the NR library. Finally, the gene sequences were aligned with the VFDB database for homology comparison using BLASTP. Figure 3The genome was mapped. Whole-genome analysis showed that the genome is dsDNA, 81715 bp in length, with a GC content of 36.74%. The genome sequence of the *Lactiplantibacillusphage* phage SAASZN03045 isolated in this invention was analyzed using BLASTN. The phage with the highest similarity to existing phages was identified as *Lactiplantibacillusphage* Gut-P1 (NCBI accession number ON117106.1), with a query cover of up to 85% and a percent identity of up to 94%. Based on a difference of more than 5%, it can be considered a novel species.

[0046] Example 2

[0047] Biological characteristics analysis of Bacterium plantarum phage SAASZN03045

[0048] 1) Host profile determination

[0049] Strawberries stored at -80℃ were streaked sequentially onto MRS solid plates and incubated statically at 37℃ for 24 hours. After single colonies grew, they were picked and transferred to MRS liquid medium and incubated overnight at 37℃. They were then stored at 4℃ for later use. 100 μL of the revived strain was evenly spread onto an MRS solid medium plate using a spreader and allowed to air dry. 1 μL of phage lysis was dropped onto the plate, 3-5 drops per plate. An equal volume of sterile physiological saline was used as a control. After air drying, the plates were placed in a constant temperature incubator and incubated at 37℃ for 24 hours. After incubation, phage zones were observed at the locations where lysis was added.

[0050] The host range of bacteriophage LJ was determined using 69 wild-type *Lactobacillus plantarum* strains screened from raw materials and fermented foods. The results are shown in Table 1. Among them, 17 *Lactobacillus plantarum* strains could be effectively lysed by bacteriophage LJ, with a lysis rate of 25%. The overall host spectrum was relatively narrow. The specific host of bacteriophages from fermented vegetables is helpful for targeted and cross-system application of the strains.

[0051] Table 1 Host spectrum of *Lactobacillus plantarum* bacteriophage SAASZN03045 in *Lactobacillus plantarum*

[0052] Note: "+" indicates that the bacteriophage can infect the bacteria; "-" indicates that the bacteriophage cannot infect the bacteria.

[0053] 2) Optimal Multiple Infection Determination

[0054] The activated host bacteria were incubated statically at 37°C until the pre-log phase, so that the concentration reached 10.7 CFU / mL; 100 μL of phage lysis buffer, 100 μL of pre-logarithmic host culture, and 4 mL of LMR-Ca liquid medium were added to MOIs of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100, respectively, and incubated at 37°C for 4 h. After incubation, each MOI sample was removed, centrifuged at 10000 rpm for 5 min, and filtered through a 0.22 μm sterile microporous membrane to obtain phage supernatants for different groups. Finally, the titer was determined using the double-layer plate method; the experiment was repeated three times, and the MOI with the highest average phage titer was considered the optimal MOI. Results are as follows: Figure 4 As shown, LJ exhibits the highest potency at an MOI of 0.1, reaching 7.74 lg (PFU / mL); and the lowest potency at an MOI of 0.0001, at 5.89 lg (PFU / mL). Therefore, the optimal MOI for LJ is 0.1.

[0055] 3) One-step growth curve determination

[0056] Centrifuge 10 mL of host bacterial culture in the pre-log phase at 8000 rpm for 5 min, discard the supernatant, and resuspend the precipitate in 1 / 4 volume of MRS-Ca liquid. Add 1 mL of phage solution at the optimal MOI to infect the bacteria, mix thoroughly, and incubate at 37°C for 30 min. Centrifuge again at 8000 rpm for 5 min to collect the bacterial pellet, wash once with the same volume of SM buffer, discard the supernatant, and repeat this step once to remove as many uninfected host free phages as possible. Resuspend the liquid in 100 mL of MRS-Ca liquid medium, mix well, and incubate at 37°C (marked as 0 min). Take samples continuously at 15 min intervals for 5 h. After centrifuging the culture medium at 5000 rpm for 10 min, use the double-layer agar method to create plaques to determine the phage titer. Plot a one-step growth curve of the phage with time on the x-axis and the logarithm of the phage titer (PFU / mL) on the y-axis. The experiment was repeated 3 times. The results are as follows: Figure 5 As shown, the phage's incubation period is approximately 30 minutes, during which phage growth is slow and it is in the incubation period, not yet released in large quantities. The lysis period is approximately 150 minutes. Between 30 and 180 minutes, the phage titer rapidly increases from approximately 1.93lg (PFU / mL) to 7.77lg (PFU / mL), initiating large-scale lysis of the host and release of progeny phages. At 180 minutes, a plateau is reached, with the phage titer remaining between 7.51 and 7.93lg (PFU / mL) for a long duration, indicating stable and sustained lysis capacity. Based on the formula (lysate yield = phage yield at the end of lysis / host cell yield at the beginning of lysis), the lysis yield of phage LJ is calculated to be approximately 59 PFU / cell.

[0057] 4) Stability testing

[0058] Temperature stability: 1 mL of phage suspension was incubated at 4℃, 20℃, 37℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour. The phage titer was determined using the double-layer plate method after incubation at different temperatures. A temperature stability curve of the phage was plotted with temperature on the x-axis and the logarithm of the phage titer (PFU / mL) on the y-axis. The experiment was repeated three times. The results are as follows: Figure 6 As shown, the phage titer remained stable within the range of 0℃ to 40℃ without significant changes, indicating that LJ has strong stability in low and medium temperature environments. When the temperature exceeds 40℃, the phage titer shows a significant decreasing trend with increasing temperature, and at 70℃ the titer drops to 0, indicating complete inactivation.

[0059] pH stability: 100 μL of phage suspension was transferred to 900 μL of SM buffer at different pH values ​​(pH 1.5-13, adjusted using NaOH and HCl), and incubated at 37°C for 1 hour. The phage titer after incubation at different pH values ​​was determined using the bilayer plate method. A pH stability curve of the phage was plotted with pH on the x-axis and the logarithm of the phage titer (PFU / mL) on the y-axis. The experiment was repeated three times. Results Figure 7 As shown, bacteriophages have strong resistance to acid and alkali, maintaining a relatively high titer within the pH range of 2 to 12, and exhibiting a wide tolerance range; however, when pH ≤ 1.5 or ≥ 13, the titer of bacteriophages drops sharply to 0, indicating that they are more sensitive to strong acid and strong alkali environments.

[0060] NaCl concentration stability: 100 μL of phage suspension was transferred to 900 μL of SM buffer with different NaCl concentrations (0%, 2%, 4%, 6%, 8%, 10%, 12%, 18%). The phage suspension was incubated at 37°C for 1 hour, and the phage titer was determined using the double-layer plate method at different pH values. A NaCl concentration stability curve was plotted with NaCl concentration on the x-axis and the logarithm of the phage titer (PFU / mL) on the y-axis. The experiment was repeated three times. The results are as follows: Figure 8 As shown, within the NaCl concentration range of 0% to 18%, the phage titer remained above 7.01g (PFU / mL), with no significant differences between the concentration groups. This high salt tolerance is consistent with its native environment, namely a salt-containing fermentation environment.

[0061] Example 3

[0062] Application of *Lactobacillus plantarum* phage SAASZN03045 in regulating the acid production process of fermented radish kimchi

[0063] I. Preparation of Radish Pickles Fermented from Bacteriophage and Compound Lactic Acid Bacteria

[0064] 1) Preparation of compound fermentation agent: *Lactobacillus plantarum* (…) Lactiplantibacillus plantarum SAASZN01137, Lactobacillus namurti ( Levilactobacillus namurensis SAASZN01047, Lactobacillus brunelli ( Lentilactobacillus buchneri SAAS-B-MRS-20200906-11, Lactobacillus brevis ( Levilactobacillus brevis SAASZN00575, ethanol-resistant Pediococcus ( Pediococcus ethanolidurans SAAS-B-MRS-20201030-3 was activated to obtain seed liquid, so that the viable count of each bacterium was 7 LogCFU / mL. The seed liquid was centrifuged to obtain bacterial precipitate. The bacterial precipitate was mixed in a ratio of 1:1:1:1:1 to obtain the compound fermentation agent.

[0065] 2) Preparation of vegetable brine mixture: Cut fresh radish into pieces, wash and drain the water, and mix with 4% brine at a ratio of 1:2 (W / V) to obtain vegetable brine mixture; 3) Inoculation and Fermentation: After inoculating the vegetable-salt mixture with a compound fermentation agent at a ratio of 2% (w / w), inoculate with *Lactobacillus plantarum* bacteriophage SAASZN03045 to achieve a titer of 10. 8 PFU / mL was used to ferment radish kimchi at 25℃ for 3 days to obtain phage + compound lactic acid bacteria group fermented radish kimchi.

[0066] Comparative experiment

[0067] Preparation of radish kimchi fermented with Lactobacillus plantarum alone, preparation of radish kimchi fermented with mixed lactic acid bacteria, and preparation of radish kimchi fermented with Lactobacillus plantarum and compound lactic acid bacteria (-)

[0068] 1) Preparation of *Lactobacillus plantarum* single-strain agent, compound lactic acid bacteria agent, and (-) *Lactobacillus plantarum* compound lactic acid bacteria agent: *Lactobacillus plantarum* (… Lactiplantibacillus plantarum SAASZN01137, Lactobacillus namurti ( Levilactobacillus namurensis SAASZN01047, Lactobacillus brunelli ( Lentilactobacillus buchneri SAAS-B-MRS-20200906-11, Lactobacillus brevis ( Levilactobacillus brevis SAASZN00575, ethanol-resistant Pediococcus ( Pediococcus ethanolidurans SAAS-B-MRS-20201030-3 was activated to obtain seed culture, and the viable count of each bacterium was 7 LogCFU / mL. The seed culture was centrifuged to obtain bacterial precipitates. The bacterial precipitates were mixed in a ratio of 1:1:1:1:1 to obtain a compound lactic acid bacteria agent; Lactobacillus namurti ( Levilactobacillus namurensis SAASZN01047, Lactobacillus brunelli ( Lentilactobacillus buchneriSAAS-B-MRS-20200906-11, Lactobacillus brevis ( Levilactobacillus brevis SAASZN00575, ethanol-resistant Pediococcus ( Pediococcus ethanolidurans The bacterial precipitate of SAAS-B-MRS-20201030-3 was mixed in a 1:1:1:1 ratio to obtain (-) Lactobacillus plantarum compound lactic acid bacteria agent; take Lactobacillus plantarum ( Lactiplantibacillus plantarum SAASZN01137 activated seed culture, viable count 7 LogCFU / mL, centrifuged to obtain bacterial precipitate as a single-strain agent of Lactobacillus plantarum.

[0069] 2) Preparation of vegetable brine mixture: Cut fresh radish into pieces, wash and drain the water, and mix with 4% brine at a ratio of 1:2 (W / V) to obtain vegetable brine mixture; 3) Inoculation and fermentation: Inoculate the vegetable brine mixture with 2% (w / w) of Bacillus plantarum single-strain fermentation agent, compound lactic acid bacteria agent, and (-) Bacillus plantarum compound lactic acid bacteria agent, respectively, and ferment at 25℃ for 3 days to obtain Bacillus plantarum fermented radish kimchi, compound lactic acid bacteria fermented kimchi, and (-) Bacillus plantarum compound lactic acid bacteria fermented radish kimchi.

[0070] Analysis of physicochemical properties, lactic acid bacteria count, organic acid composition, and volatile flavor components during the fermentation process of radish kimchi fermented with bacteriophage + compound lactic acid bacteria, Lactobacillus plantarum, compound lactic acid bacteria, and (-) Lactobacillus plantarum and compound lactic acid bacteria.

[0071] 1) Physicochemical index determination: The reducing sugar content in the sample was determined using the 3,5-dinitrosalicylic acid method (DNS method). 1 mL of fermentation broth sample was placed in a 25 mL stoppered colorimetric tube, and 2 mL of DNS reagent was added and thoroughly mixed. The tube was then placed in a 100℃ water bath for 3 minutes. After the time was reached, it was quickly removed and allowed to cool naturally to room temperature before being diluted to the mark. Finally, 200 μL of the reaction solution was taken and its absorbance was measured at 540 nm using an ELISA reader. Referring to the pH meter potentiometric titration method in GB12456-2021 "National Food Safety Standard - Determination of Total Acid in Food (including Amendment No. 1)," the total acid content of the fermentation broth sample was determined. Specifically, 3 mL of fermentation broth sample was mixed thoroughly with 50 mL of distilled water, and then titrated with 0.1 mol / L NaOH standard solution. Titration was stopped when the pH reached 8.2. The volume of NaOH standard solution consumed was recorded and calculated using the lactic acid conversion coefficient. The pH value was determined using a pH meter in accordance with GB5009.237-2016, "National Food Safety Standard - Determination of pH Value in Food".

[0072] 2) Lactic acid bacteria count: The lactic acid bacteria count was determined in accordance with GB4789.35—2023 "National Food Safety Standard for Microbiological Examination of Food - Lactic Acid Bacteria Examination", that is: the sample was diluted 10 times and spread on MRS medium under aseptic conditions and incubated at 37℃ for 3 days under anaerobic conditions.

[0073] 3) Determination of Organic Acid Content: Take 2 mL of sample, centrifuge at 10000 rpm for 10 min, collect the supernatant, and then filter it through a 0.22 μm filter membrane. The filtrate is then analyzed qualitatively and quantitatively for organic acid components using high-performance liquid chromatography (HPLC). The mobile phase used is 0.005 mol / L sulfuric acid solution, the column flow rate is set to 0.6 mL / min, and signal acquisition is performed at 215 nm using a UV detector. The column temperature is controlled at 35℃, and the complete detection cycle for a single sample is set to 30 min. After the detection is completed, the retention time data from the HPLC chromatogram is used for qualitative identification of organic acids, and the specific contents of lactic acid, acetic acid, succinic acid, malic acid, and citric acid in the sample are determined using peak area quantitative analysis.

[0074] 4) Determination of volatile flavor compounds: The volatile compounds in the juice of fermented radish pickles were analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). 5 mL of sample was accurately measured, and 20 μL of methyl octanoate internal standard at a concentration of 73 μg / mL was added to a 20 mL headspace vial for GC-MS analysis. The instrument settings were as follows: HP-5MS column was used. UI type (size 30m×0.25mm×0.25μm); high-purity helium (He) as carrier gas, flow rate maintained at 1.0mL / min, using automatic splitless injection mode; temperature program: initial temperature 40℃, hold for 5min, then increase to 100℃ at a rate of 4℃ / min, then increase to 250℃ at a rate of 6℃ / min and hold for 10min; mass spectrometry detection uses electron ionization (EI) source, electron energy 70eV, mass transfer line and ion source temperature both set to 250℃, scan mass-to-charge ratio range 35-550m / z; for qualitative analysis, the measurement results are compared with the 2001 standard spectral library of the National Institute of Standards and Technology (NIST), and substances with a matching degree greater than or equal to 75 are screened. The qualitative confirmation of volatile substances is completed by combining the comparison results of mass spectra and standard spectra, retention index and relevant literature reports; for quantitative analysis, the relative content of each substance is calculated by area normalization method, and semi-quantitative analysis is carried out by internal standard method.

[0075] The results of the physicochemical parameters of reducing sugar, total acid and pH are as follows: Figures 9-11As shown, the results indicate that compared to the *Lactobacillus plantarum* group, the (-) *Lactobacillus plantarum* combined with lactic acid bacteria fermentation of radish kimchi, and the combined lactic acid bacteria group, the bacteriophage + combined lactic acid bacteria group consumed reducing sugars faster and produced total acid more quickly, resulting in a lower pH; the lactic acid bacteria count results are shown in the figure. Figure 12 As shown, the results indicate that the number of viable lactic acid bacteria in the phage + compound lactic acid bacteria group is slightly higher than that in the other three groups (the phage in this invention has a narrow lysis range, only infecting *Lactobacillus plantarum* in the compound lactic acid bacteria group of this invention; therefore, this combination allows for more niches for other lactic acid bacteria after the phage lyses the host bacteria, resulting in better growth and a slightly higher number of bacteria compared to the other three groups); combined with the organic acid composition ( Figure 13 This indicates that the slightly higher number of lactic acid bacteria in the phage + complex lactic acid bacteria group accelerated the production of organic acids, especially the accumulation of lactic acid, which has a milder sour taste, while reducing the content of acetic acid, which has a more pungent sour taste; the volatile component composition results are as follows: Figure 14 As shown, the results indicate that the Lactobacillus plantarum group, the (-) Lactobacillus plantarum compound lactic acid bacteria fermented radish kimchi and the compound lactic acid bacteria group contain more sulfur-containing compounds and nitriles that present a pungent and spicy taste, while the bacteriophage + compound lactic acid bacteria group promotes the formation of volatile acids, indicating that the bacteriophage + compound lactic acid bacteria group alleviates the pungent and spicy taste and promotes the rapid formation of a mellow sour and fragrant flavor.

[0076] As can be seen from the above embodiments, the plant lactobacillus phage SAASZN03045 provided by the present invention has high specificity, which is beneficial to maintaining the balance of fermentation flora. At the same time, it can promote the rapid production of lactic acid by flora in fermented vegetables, reduce the content of acetic acid, and enhance the sour and fragrant flavor. This phage is adapted to the fermentation vegetable production environment and can solve the industrial problems of long fermentation cycle, slow acid production and insufficient aroma in fermented vegetables. It has a wide range of application scenarios and market development potential.

[0077] 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 plant lactobacillus phage ( Lactiplantibacillus plantarum phage)SAASZN03045, characterized in that, The preservation number of the plant lactobacillus phage SAASZN03045 is GDMCC NO:67684-B1.

2. The *Lactobacillus plantarum* bacteriophage SAASZN03045 according to claim 1, characterized in that, The titer of the plant lactobacillus phage SAASZN03045 was 10. 8 ~10 9 PFU / mL.

3. The *Lactobacillus plantarum* bacteriophage SAASZN03045 according to claim 1, characterized in that, The physicochemical stability range of the plant lactobacillus phage SAASZN03045 is as follows: temperature 0℃~40℃, pH value 2~12, NaCl concentration 0%~18%.

4. The application of the plant lactobacillus phage SAASZN03045 according to any one of claims 1 to 3 in regulating plant lactic acid bacteria fermentation of vegetables.

5. A method for preparing fermented vegetables, characterized in that, Includes the following steps: After inoculating the vegetable brine mixture with a compound fermentation agent, inoculate it with the plant lactobacillus phage SAASZN03045 as described in any one of claims 1 to 3, and ferment at 20 to 25°C for 2 to 5 days to obtain fermented vegetables.

6. The preparation method according to claim 5, characterized in that, The compound fermentation agent comprises strains in the following mass ratio: *Lactobacillus plantarum* SAASZN01137: *Lactobacillus namurti* SAASZN01047: *Lactobacillus bruneri* SAAS-B-MRS-20200906-11: *Lactobacillus brevis* SAASZN00575: *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 in a ratio of (1~1.2):(0.8~1):(0.9~1.1):(1~1.2):(0.1~1); the viable cell concentrations of *Lactobacillus plantarum* SAASZN01137, *Lactobacillus namurti* SAASZN01047, *Lactobacillus bruneri* SAAS-B-MRS-20200906-11, *Lactobacillus brevis* SAASZN00575, or *Pediococcus ethanol-resistant* SAAS-B-MRS-20201030-3 are 10... 6 ~10 8 CFU / mL.

7. The preparation method according to claim 6, characterized in that, The inoculum amount of the compound fermentation agent is 2%~5% (w / w).

8. The preparation method according to claim 5, characterized in that, The titer of the plant lactobacillus phage SAASZN03045 was 10. 8 ~10 9 PFU / mL.

9. The preparation method according to claim 5, characterized in that, The ratio of vegetables to brine in the vegetable-salt mixture is 1g:(1~3)mL, and the salt content of the brine is 1%~8%.

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