Cracking lactobacillus plantarum phage and application thereof

Abstract

The invention belongs to the technical field of fermentation engineering, and discloses a lytic lactobacillus plantarum phage and application thereof. The lactobacillus plantarum phage ZMDQ02 is obtained through screening, the lactobacillus plantarum phage ZMDQ02 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the lactobacillus plantarum phage ZMDQ02 is CGMCC NO.46160. According to the lactobacillus plantarum phage ZMDQ02, the lactobacillus plantarum phage ZMDQ02 can be used for preparing the lactobacillus plantarum phage ZMDQ02. The bacteriophage is applied to the culture process of high-temperature yeast, has obvious influence on volatile flavor substances of the high-temperature yeast, can endow the yeast with more typical flavor and thicker yeast flavor, and has better research and development value and application prospect in the field of white spirit brewing. According to the method, the high specificity of the phage technology is combined with the complex micro-ecology of the traditional fermentation process, the microflora structure of the yeast for making hard liquor can be optimized, the metabolite spectrum is improved, and finally the flavor quality of the white spirit is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a lytic Lactobacillus plantarum bacteriophage and its applications, particularly in the production of high-temperature Daqu (a type of starter culture). Background Technology

[0002] High-temperature Daqu is a traditional Chinese brewing fermentation agent widely used in the production of Baijiu (Chinese liquor). Its production typically involves pure wheat (or barley, peas) through solid-state fermentation under the influence of a natural microbial community. The process includes six main steps: blending raw materials according to a specific ratio, adding water and then crushing, mixing, and pressing to form koji blocks. These blocks are then placed in a fermentation room for solid-state cultivation and maturation, ultimately resulting in mature Daqu. Based on the highest core temperature caused by the activity of the natural microbial community during cultivation, Daqu can be classified into high-temperature Daqu (60–70℃), medium-temperature Daqu (50–60℃), and low-temperature Daqu (40–50℃). Daqu with a maximum core temperature between 60 and 70℃ is called high-temperature Daqu. High-temperature Daqu employs an open, complex microbial community fermentation model, resulting in an extremely complex microbial community structure, primarily including bacteria (such as lactic acid bacteria and Bacillus), molds (such as Rhizopus and Aspergillus), yeasts, and actinomycetes. The interactions between these microorganisms directly affect the enzyme composition and metabolite profile of the Daqu, thus determining the final quality and flavor characteristics of the Baijiu product. There are currently few reports on how to give Daqu (a type of Chinese liquor) its unique flavor. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, the present invention provides a lytic Lactobacillus plantarum phage and its application.

[0004] In a first aspect of the present invention, a lytic Lactobacillus plantarum phage strain ZMDQ02 is provided, which was deposited on November 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46160.

[0005] Specifically, the bacteriophage ZMDQ02 is a long-tailed bacteriophage with an icosahedral capsid structure. Transmission electron microscopy revealed that the phage consists of a head of (53.2±4.7)×(46.7±4.2) nm and a long, non-contracting tail of 330.6±20.7 nm. The complete genome of bacteriophage ZMDQ02 is linear dsDNA with a genome length of 26282 bp and a C+G content of 39.62%. The optimal multiplicity of infection (MOI) is 0.0001, at which the phage exhibits the highest efficiency in infecting the host and releases the largest number of progeny phages, reaching up to 10. 9 PFU / mL.

[0006] In a second aspect of the invention, a kit is provided comprising the phage ZMDQ02 described in the first aspect.

[0007] Specifically, the kit also includes a preservation solution, such as SM buffer, and is stored at 4°C.

[0008] In a third aspect of the invention, a bacteriophage preparation is provided, comprising the bacteriophage ZMDQ02 described in the first aspect and Lactobacillus plantarum.

[0009] Specifically, the potency ratio of bacteriophage ZMDQ02 to Lactobacillus plantarum is 1:1.

[0010] Specifically, in the formulation, bacteriophage ZMDQ02 and Lactobacillus plantarum are each stored separately.

[0011] Specifically, the Lactobacillus plantarum is one that can be used in the food industry, such as any suitable commercially available Lactobacillus plantarum strain or a host bacteriophage preserved together with bacteriophage ZMDQ02.

[0012] In a fourth aspect of the invention, the application of the phage ZMDQ02 described in the first aspect, the kit described in the second aspect, and the phage preparation described in the third aspect in the production of Daqu (especially high-temperature Daqu) and Baijiu (e.g., sauce-flavored and strong-flavored Baijiu).

[0013] Specifically, the bacteriophage ZMDQ02 described in this invention is used in the production of Daqu (a type of starter culture), which can enable Daqu to produce more flavor compounds.

[0014] In some embodiments of the present invention, the Daqu is high-temperature Daqu (Daqu with the highest core temperature between 60-70°C) or medium-high temperature Daqu (Daqu with the highest core temperature between 50-70°C).

[0015] In some embodiments of the present invention, the liquor is a sauce-flavored liquor.

[0016] In a fifth aspect of the present invention, a method for producing Daqu (a type of starter culture) is provided, comprising the steps of adding the bacteriophage ZMDQ02 described in the first aspect or the bacteriophage preparation described in the third aspect to pulverized Daqu raw materials, and then cultivating the Daqu.

[0017] Specifically, the raw materials for the Daqu (a type of starter culture) are selected from one or more of wheat, barley, peas, etc., especially wheat.

[0018] Specifically, the potency ratio of the amount of bacteriophage added to the amount of Lactobacillus plantarum added was 1:1.

[0019] Specifically, after adding the bacteriophage ZMDQ02 described in the first aspect or the bacteriophage preparation described in the third aspect to the pulverized Daqu raw material, the method further includes the step of pressing the resulting raw material mixture into Daqu blocks.

[0020] Specifically, the method further includes the step of adding mother koji to the pulverized koji raw material.

[0021] In some embodiments of the present invention, the raw material for the Daqu (a type of starter culture) is wheat, and the method includes the following steps: (1) Moistening wheat: Adding water to wheat; (2) Grinding: Grind the wheat obtained in step (1) according to the standard of "rotten heart but not rotten skin" and "plum petal", or according to the standard of 40 mesh sieve, with the ratio of upper grains to lower powder being 1:1. (3) Mixing and pressing: Add water, mother koji, and bacteriophage ZMDQ02 (or the bacteriophage preparation mentioned above in this invention, adding Lactobacillus plantarum can further exert the effect of bacteriophage and enrich the metabolites in koji) to the pulverized raw materials obtained in step (2) (mix evenly, the mixing standard is "the mixture can be kneaded into a ball and does not stick to the hand"), put it into a mold, and press it into shape; (4) Cultivation: Cultivate and ferment the koji blocks obtained in step (3).

[0022] More specifically, the water added in steps (1) and (3) is sterile water, especially sterile deionized water.

[0023] More specifically, the proportion of water added in step (1) is 1%-10% of the wheat (volume mass ratio, v / w, mL / g), for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, especially 4%-6%, 5%.

[0024] More specifically, the soaking time in step (1) is 8-24 hours, for example 10, 12, 14, 16, 18, 20, 22, 24 hours.

[0025] More specifically, step (1) includes: adding water to the wheat, mixing well, and letting it sit overnight.

[0026] More specifically, the proportion of water added in step (3) is 35%-45% (v / w, mL / g) of wheat, for example 35%, 36%, 38%, 39%, 40%, 41%, 42%, 44%, 45%, especially 38%-42%, 40%.

[0027] More specifically, the proportion of mother koji added in step (3) is 5%-20% of the wheat (by weight, w / w), for example 5%, 6%, 8%, 9%, 10%, 11%, 12%, 14%, 15%, 16%, 18%, 20%, especially 8%-12% and 10%. The mother koji can be any suitable commercially available high-temperature koji.

[0028] More specifically, based on the amount of wheat, the inoculation amount of *Lactobacillus plantarum* in step (3) is 0.5%-5% (v / w) (e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%), and the amount of bacteriophage added is based on a multiplicity of infection of 1-5 (e.g., 1, 2, 3, 4, 5). In some embodiments of the present invention, the inoculation amount of *Lactobacillus plantarum* is 1%, and the multiplicity of infection of the bacteriophage is 1.

[0029] More specifically, the core temperature for bacterial culture and fermentation in step (4) is 35-65℃, for example 40, 42, 44, 45, 46, 48, 50, 52, 54, 56, 58, 60℃, 65℃, especially 50-65℃.

[0030] More specifically, the humidity of the bacterial culture and fermentation in step (4) is 45%-80%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%.

[0031] More specifically, the fermentation time described in step (4) is 10-60 days, for example, 10, 20, 30, 40, 50, or 60 days.

[0032] In a seventh aspect of the invention, a large-grain liquor produced by the method described in the sixth aspect is provided.

[0033] In an eighth aspect of the present invention, a method for producing baijiu (Chinese liquor) is provided, comprising the step of fermentation using the daqu (a type of starter culture) described in the seventh aspect.

[0034] In a ninth aspect of the present invention, a type of liquor produced by the method described in the eighth aspect is provided.

[0035] Many strains of lactic acid bacteria have applications in the food industry, such as *Lactobacillus*, *Lactococcus*, *Leuconostoc*, and *Pediococcus*, playing a crucial role in fermented foods like dairy products, pickled vegetables, beer, and spirits. These strains are classified as "probiotics," potentially promoting gut microbiota health and consumer health. Furthermore, the application of well-defined native microorganisms during fermentation is a powerful method to improve product quality and safety. For example, in cheese manufacturing, adding selected lactic acid bacteria strains can alter or improve aroma by increasing the concentration of branched-chain acids and ethyl esters.

[0036] Lactobacillus plantarum is widely distributed in many natural environments, especially in various fermented foods. Studies have screened species of Lactobacillus plantarum from Daqu (a type of starter culture) for alcoholic fermentation, finding a significant increase in flavor compounds such as ethyl acetate, ethyl lactate, tetramethylpyrazine, and 4-ethyl-2-methoxyphenol. This enhances flavor, controls the quality of Baijiu (Chinese liquor), and allows for the construction of synthetic microbial phages for Daqu. These phages are widely distributed in the natural environment and can drive the regulation of microbial community structure and ecosystem function.

[0037] Bacteriophage-bacterial interactions are crucial for microbial evolution, potentially influencing the balance between different microbial populations, including community composition, species evolution, and interspecies interactions, thereby reshaping microbial communities. Currently, bacteriophages have been discovered and isolated in the production of various fermented foods, including fermented soybeans, fermented soy products, fermented dairy products, fermented vegetables, and fermented alcoholic beverages. Numerous studies have shown that bacteriophages can harm the fermentation process. The presence of bacteriophages, the main functional bacteria in fermentation, typically inhibits the normal growth of host bacteria or causes massive lysis of host bacteria, leading to abnormal fermentation or even failure. On the other hand, bacteriophages can specifically lyse pathogenic bacteria or bacteria that produce undesirable flavors, having a beneficial effect on fermentation and achieving good biocontrol. Some studies have also found that certain sequences of bacteriophages from fermenting bacteria can help host bacteria autolyze, releasing intracellular enzymes and improving the flavor of fermented foods. Furthermore, screening for hosts insensitive to bacteriophages can, to some extent, prevent fermentation failure and influence community succession, thus helping to advance the fermentation process. Bacteriophages have become an emerging biological control tool due to their high host specificity. However, existing research on the application of bacteriophages mainly focuses on the field of food safety (such as controlling pathogenic bacteria), and there are no reports on the precise regulation of functional microorganisms (such as lactic acid bacteria) during fermentation.

[0038] The inventors of this invention have screened and obtained a strain of *Lactobacillus plantarum* phage, which has the following advantages: high titer, strong lytic ability, high lytic specificity, high temperature tolerance, and wide acid-base tolerance range, which is beneficial for industrial applications. In particular, applying this phage to the cultivation process of high-temperature and medium-high-temperature koji (a type of Chinese liquor) can regulate the microbial community structure during fermentation, significantly affecting the volatile flavor compounds of high-temperature and medium-high-temperature koji, and imparting typical aromas such as soy sauce aroma, fermented black bean aroma, and roasted aroma to the koji. It has excellent research and development value and application prospects in the field of baijiu (Chinese liquor) brewing.

[0039] This invention combines the high specificity of bacteriophage technology with the complex microecology of traditional fermentation processes to optimize the microbial community structure of Daqu (a type of starter culture), thereby improving the metabolite profile and ultimately enhancing the flavor quality and stability of Baijiu (Chinese liquor) products. Attached Figure Description

[0040] Figure 1 A schematic diagram (A) of phage plaques of ZMDQ02 on a double-layer agar plate based on the "drop method" and the "plaque method" respectively, and a transmission electron microscope image (B) of phage ZMDQ02.

[0041] Figure 2 The diagram shows the genome of bacteriophage ZMDQ02.

[0042] Figure 3The figure shows the optimal infection multiplicity for bacteriophage ZMDQ02.

[0043] Figure 4 The figure shows the one-step growth curve of bacteriophage ZMDQ02.

[0044] Figure 5 The temperature stability of bacteriophage ZMDQ02 is shown.

[0045] Figure 6 The acid-base stability of bacteriophage ZMDQ02 is shown.

[0046] Figure 7 The image shows the effect of bacteriophage ZMDQ02 on the production of lactic acid by the host fermentation.

[0047] Figure 8 The changes in the temperature (A) of the koji core and the humidity (B) of the koji room during the koji cultivation process in the distillery were used as simulated koji cultivation conditions.

[0048] Figure 9 The figure shows a comparison of the composition and content of volatile substances in different treatment groups of Daqu in Example 9.

[0049] Figure 10 The image shows a heatmap of the content of characteristic flavor compounds in high-temperature Daqu (a type of starter culture) in Example 9. Detailed Implementation

[0050] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0052] According to the "List of Microbial Strains that Can Be Used in Food" and the "List of Microbial Strains that Can Be Used in Infant Food" updated by the National Health Commission in 2022, Lactiplantibacillus plantarum and Lactobacillus plantarum represent the same microbial species, and the two can be used interchangeably in this invention.

[0053] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0054] In this invention, v / w represents the volume-to-mass ratio, expressed in mL / g. For example, adding 5% (v / w) water to wheat means adding 5 mL of water per 100 g of wheat. w / w represents the mass ratio. For example, adding 10% (w / w) mother koji (based on the mass of wheat) means adding 10 g of mother koji per 100 g of wheat.

[0055] Example 1: Isolation and screening of bacteriophages 1. Collection and processing of high-temperature Daqu samples Take 5 g of high-temperature Daqu sample (taken from a traditional sauce-flavored Baijiu distillery in Zunyi City, Guizhou Province), mix with 15 mL of AKC buffer (modified 1% potassium citrate solution, containing 1% potassium citrate, 10% 1×PBS, 5 mM EDTA and 150 mM MgSO4), and shake at 30℃ and 400 rpm for 1 h. Vortex the mixture for 1 min every 20 min to desorb the virus. Centrifuge (500 g, 4℃) for 5 min to remove large particles. The resulting supernatant is the treated Daqu extract.

[0056] 2. Enrichment and purification of bacteriophages Add 2% MRS broth powder and 10 mM calcium chloride to the Daqu extract and incubate overnight at 30°C. Filter the solution through 0.45 and 0.22 μm membranes for sterilization; the filtrate is the phage proliferation solution.

[0057] The typical "drop method" and "plaque method" are used to verify whether the phage proliferation solution contains phages. If the drop liquid forms a single "plaque" on the upper plate containing the host bacteria, it is a phage plaque, which proves that the proliferation solution contains the phage of the host bacteria.

[0058] The specific steps are as follows: Use a pipette tip or inoculation loop to directly pick up plaques into 1 mL of SM buffer, let stand for 5-10 minutes, then centrifuge (8000 g, 4℃) for 10 minutes. Dilute 10-fold or 100-fold with SM buffer, generally to 100-fold. -5 -10 -6 Mix 100 μL of diluent with 100 mL of bacterial culture, let stand for 15 min, then spread on a double-layer plate. Repeat the purification process 3-5 times until the phage plaques are of uniform size and transparency, thus completing the phage purification.

[0059] 3. Mass amplification and concentration of bacteriophages Inoculate 2 mL of overnight cultured host bacteria into 200 mL of MRS broth and incubate until OD500. 600Add 1 mL of purified phage to a concentration of 0.3-0.5 mL and incubate overnight at 30°C to allow complete lysis of the host bacteria. Centrifuge (10,000 g, 4°C) for 20 min and collect the supernatant. Add sodium chloride to a final concentration of 1 M, incubate on ice for 1-2 h, centrifuge (10,000 g, 4°C) for 20 min, and collect the supernatant. Add PEG-8000 (10 g / 100 mL) and gently stir to dissolve, incubate on ice overnight to precipitate the phage. Centrifuge (10,000 g, 4°C) for 20 min, discard the supernatant, and invert the centrifuge tube for 5 min to allow the residual liquid to drain completely. Resuspend the precipitate in 1 mL of SM buffer, gently pipetting from top to bottom. Extract with an equal volume of chloroform, shake for 30 s, centrifuge (5000 g, 4℃) for 15 min, recover the upper aqueous phase containing phage, add an equal volume of chloroform for extraction, repeat 3-5 times until the upper liquid phase is clear, and obtain the recovered phage concentrate, which is stored at 4℃ for later use.

[0060] 4. Phage screening and isolation A bacteriophage strain was obtained from Daqu (a type of starter culture) through spot testing. This phage exhibited lytic characteristics against *Lactobacillus plantarum*, and distinct vacuoles (such as those observed on double-layer plates) were observed. Figure 1 (As shown).

[0061] 5. Preservation of bacteriophages The bacteriophage was named Lactobacillus plantarum phage ZMDQ02 (hereinafter referred to as "ZMDQ02" in this invention), and it was deposited together with the host bacterium at the China General Microbiological Culture Collection Center on November 6, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.46160.

[0062] Example 2: Electron microscopic observation of bacteriophages Use valence greater than 10 7 The phage concentrate at PFU / mL was used as the sample to be observed. 10 μL of the phage concentrate was spotted onto a copper mesh, allowed to stand for 1 min, blotted dry with filter paper, and then a drop of phosphotungstic acid staining solution was added. After staining for 1 min, the solution was blotted dry with filter paper and observed on the instrument.

[0063] like Figure 1 As shown, bacteriophage ZMDQ02 has an icosahedral capsid structure with a head size of (53.2±4.7)×(46.7±4.2) nm and a tail sheath length of 330.6±20.7 nm.

[0064] Therefore, this bacteriophage belongs to the order Caudovirales and the family Siphoviridae, based on its morphological classification.

[0065] Example 3: Extraction of bacteriophage DNA and whole-genome sequencing and analysis Use valence greater than 10 7 PFU / mL phage concentrate was used as the sample to be extracted. Phage DNA was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0. The whole genome sequencing of the phage was completed by technical services provided by Megagene Corporation. The company mainly uses second-generation whole genome sequencing technology. The specific operation is as follows: (1) Library preparation: First, the DNA sample is randomly fragmented to generate DNA fragments of the required length. The sticky ends formed by the fragmentation are repaired into blunt ends. Then, the 3' end is added with the base "A" so that the DNA fragment can be linked with the special adapter with the "T" base at the 3' end. Finally, the DNA fragment with adapters at both ends is amplified by PCR technology to complete the construction of the entire library. The qualified library is prepared for cluster preparation and sequencing. (2) Sequencing was performed on the machine; (3) The quality control process used Soapnuke software for data filtering and BWA software to remove host contamination; (4) Megahit was used to assemble high-quality reads of each sample to obtain contig sequence splicing, and finally the phage sequence was obtained.

[0066] The BLASTn tool (https: / / blast.ncbi.nlm.nih.gov / Blast) was used to assess the novelty of bacteriophages' closely related species and sequences in the NCBI Virus Database. Genomic functional protein sequence prediction, and the prediction and annotation of putative open reading frames (ORFs) were performed and plotted in CGView using PHAGEST to complete the bacteriophage's whole genome map. Evolutionary relationship analysis of the bacteriophages primarily involved comparing conserved terminal large subunit enzyme sequences with 20 closely related bacteriophages, and the analysis was performed using MEGA7 software. The putative tRNA coding genes, the Virulence Factor Database (VFDB), and the Antibiotic Resistance Gene Database were used to assess the safety of the bacteriophages.

[0067] like Figure 2As shown, the complete genome of bacteriophage ZMDQ02 is linear dsDNA with a length of 26282 bp and a C+G content of 39.62%. In the NCBI database, the strain with the highest genome similarity to bacteriophage ZMDQ02 is a Salmonella bacteriophage from metagenomics data (the reference sequence BK044213.1 in the database matches the ZMDQ02 sequence by 709 bp, with a similarity of 94.076%). According to the latest classification rules of the International Committee on Taxonomy of Viruses (ICTV), if the genome differences between two viruses exceed 5%, they are classified as different species; if the differences exceed 30%, they are classified as different genera. Therefore, bacteriophage ZMDQ02 can be defined as a novel bacteriophage. Thus, the bacteriophage ZMDQ02 proposed in this invention possesses research value for species novelty and can enrich the currently known bacteriophage library worldwide.

[0068] PHASGEST annotation of the entire genome of the bacteriophage revealed 34 known ORFs for ZMDQ02, including terminator proteins, portal proteins, proteases, capsid proteins, and tail-related proteins. The remaining genes were defined as hypothetical proteins, and all genes were located on the positive strand. Furthermore, no antibiotic resistance genes or virulence factor-related genes were found, indicating that this bacteriophage possesses the safety for application at the genetic level.

[0069] Example 4: Optimal Multiple of Infection (MOI) of bacteriophages The optimal MOI for bacteriophage ZMDQ02 was determined using the double-layer plate method. The optical density of the host bacteria in the early logarithmic phase was adjusted to approximately OD using fresh MRS broth. 600 =0.3 (bacterial concentration is approximately 10) 8 (CFU / mL). Host bacterial culture and phage serial dilutions were mixed at multiples of infection (MOI) of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, and incubated overnight at 30°C in 10 mL of MRS broth. The phage titer was determined, and the highest MOI was defined as the optimal MOI. Three parallel experiments were conducted for each group.

[0070] The results are as follows Figure 3 As shown, although ZMDQ02 achieved optimal infection at MOI=0.0001, its titer was not statistically different from that of the treatment groups with MOIs of 0.001 and 0.01.

[0071] Example 5: One-step growth curve of bacteriophage The optical density of the host bacteria in the early logarithmic stage was adjusted to approximately OD using 5 mL of fresh MRS broth. 600=0.3 (bacterial concentration is approximately 10) 8 The bacterial cells were collected by centrifugation (10,000 g, 4℃) for 5 min at CFU / mL. The precipitate was resuspended in 1 mL of MRS broth and mixed with 1 mL of phage (using the optimal multiplicity of infection determined above). After standing for 5 min to allow adsorption, the mixture was centrifuged (10,000 g, 4℃) for 5 min. The supernatant was discarded, and the centrifuge tube was inverted to allow the residual liquid to drain completely, thus removing unadsorbed phage. The precipitate was resuspended in 5 mL of MRS broth and incubated at 30℃. Samples were taken every 10 min to determine the titer. Three parallel experiments were set up for each group, and the average value was used to plot a one-step growth curve.

[0072] The results are as follows Figure 4 As shown, ZMDQ02 has a relatively long latency and lysis period, both around 90 min, and the average burst rate of ZMDQ02 is 91 PFU / cell.

[0073] Example 6: Temperature stability of bacteriophages Take 1 mL of phage proliferation solution (potency approximately 10). 8 Divide the phage (PFU / mL) into several portions in 1.5 mL sterile centrifuge tubes and place them in constant temperature water baths at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1 hour each. After the time is up, immediately transfer the centrifuge tubes to an ice box and determine the phage titer. The temperature stability of the phage can be assessed based on the change in titer. Three parallel experiments were set up for each group.

[0074] like Figure 5 As shown, ZMDQ02 did not show significant changes in potency after incubation at 30-40℃ for 1 h (P>0.5). At 50-60℃, its potency decreased by 2 lg PFU / mL, and at 70℃ it decreased to 140 PFU / mL. It was completely inactivated at 80℃.

[0075] Example 7: pH stability of bacteriophages The pH of the SM buffer solution was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using sodium hydroxide and hydrochloric acid solutions, respectively. 100 μL of phage proliferation medium (potency approximately 10) was then used. 8 The phage titer was determined by mixing the phage (PFU / mL) with 900 μL of the above broth culture medium and incubating at 4°C for 1 h. The pH stability of the phage could be assessed based on the change in titer. Three parallel experiments were conducted for each group.

[0076] like Figure 6As shown, ZMDQ02 is poorly tolerant to acidic environments with pH < 4 and will be completely inactivated after 1 h of treatment. However, its potency is relatively stable (> 8 lg PFU / mL) under pH 4-10 conditions. It can also tolerate strongly alkaline environments, and its potency is stable at around 6.5 lg PFU / mL at pH 11-12.

[0077] Example 8: Bacteriophage Fermentation Experiment Bacteriophage fermentation was performed using MRS broth, and lactic acid content was measured to characterize the effect of bacteriophages on lactic acid production by host lactic acid bacteria. Overnight cultured bacterial suspensions were inoculated into three groups of 200 mL MRS broth and cultured until OD... 600 The concentrations were 0.3-0.5 mg / L. One group served as the control group, receiving buffer solution. The remaining two groups received phage at MOIs of 0.0001 and 1, respectively, designated as the low MOI and high MOI groups. Samples were collected every 12 hours until 72 hours, and lactate levels were determined using a lactate content assay kit (visible spectrophotometry). Each group was tested in triplicate.

[0078] like Figure 7 As shown, after the addition of bacteriophage ZMDQ02, the lactic acid content in the low MOI group increased significantly between 12 and 36 h, even exceeding that of the control group. This may be due to the emergence of resistant bacteria caused by the addition of a small amount of bacteriophage. At the end of fermentation, there was no significant difference in lactic acid content between the control group and the low MOI group. Compared with the other two groups, the high MOI group did not show a significant increase in lactic acid content between 12 and 24 h, possibly due to the lysis of the host bacteria by the bacteriophage. However, after 24 h, the lactic acid content began to increase rapidly, reaching a peak at 48 h and then decreasing significantly. Throughout the process, the lactic acid content in this group remained lower than that in the other two groups, indicating that only high concentrations of ZMDQ02 had a significant inhibitory effect on the metabolism of Lactobacillus plantarum.

[0079] Example 9: Effect of adding bacteriophage ZMDQ02 on the flavor of high-temperature Daqu.

[0080] The process of cultivating high-temperature koji (a type of starter culture) in a distillery was simulated. Environmental parameters for the simulated koji-making process were adjusted based on actual temperature and humidity monitoring data from the distillery's koji-making room. Bacteriophages were added during the koji-mixing process. The process flow and temperature and humidity parameters for the simulated koji-making experiment were set with reference to monitoring data from the distillery's koji-making room (see [link to relevant documentation]). Figure 8 (Three parallel tests were conducted at each time point). The process flow for producing high-temperature Daqu (a type of Chinese liquor) is as follows: (1) Wheat conditioning: Add 5% (volume mass ratio, v / w, mL / g) of sterile deionized water to wheat, mix well, and let stand overnight; (2) Crushing: Grind wheat to the standard of "rotten heart but not rotten skin" and "plum petal", or to the standard of 40 mesh sieve, with the ratio of upper grains to lower flour being 1:1; (3) Mixing and pressing: Based on the weight of wheat, add 40% water (v / w) and 10% mother koji (commercially available) (mass ratio, w / w), mix evenly, and the mixing standard is "it can be kneaded into a ball by hand without sticking to the hand", put it into the mold and press it into shape; Cultivation: Place the koji blanks in an incubator for cultivation.

[0081] This part of the experiment is mainly divided into three groups: blank group, control group, and experimental group. Bacteriophages were added or not added during the mixing step, as shown in the table below. All treatment groups maintained the same amount of water added during mixing, and all other processes were completely identical.

[0082] Table 1 Different treatment groups Note: The Lactobacillus plantarum used in this experiment is a host bacterium preserved together with bacteriophage ZMDQ02.

[0083] Samples cultured for 40 days were subjected to GC-MS analysis. The center and edge portions of each Daqu (a type of starter culture) were selected, pulverized, and placed in sterile bags. All samples were stored at -80℃ for analysis after collection. The volatile compound components and contents of different sample groups were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HSP-MS). The specific procedures are as follows: Sample pretreatment: Accurately weigh 5 g of Daqu sample, add 20 mL of ultrapure water and sonicate for 30 min to obtain supernatant; add 1.5 g of sodium chloride to a 20 mL headspace vial with a silicone diaphragm seal, and add 8 mL of the collected supernatant and 10 µL of internal standard (20 mg / L 2-octanol) together.

[0084] GC / MS settings: Aged 50 / 30 µm DVB / CAR / PDMS fibers were inserted into vials and adsorbed at 40 °C for 30 min. After headspace sampling, separation and detection were performed using a GC-Q / TOF-MS system equipped with a DB-WAX (30 m × 250 µm × 0.25 µm) capillary column. Pure helium was used as the carrier gas at a flow rate of 1 mL / min. SPME extracts were injected in splitless mode. The injection port temperature was 250 °C, and the thermal desorption time was 5 min. The temperature program was as follows: 40 °C held for 1 min, then increased to 100 °C at a rate of 2.5 °C / min, held for 1 min, then increased to 160 °C at 3 °C / min without holding; then increased to 230 °C at 5 °C / min and held for 20 min. Mass spectra were recorded under electron impact from a 70 eV ion source (EI). The ion source and quadrupole temperatures were 230 °C and 150 °C, respectively. The mass spectrometry scanning range is 25~500 amu.

[0085] After obtaining the data from the instrument, the metabolomics data were systematically analyzed using MSdial software (version 4.9). In positive ion mode, various adduct ions, including [M+H]+, [M+NH4]+, [M+K]+, [M+Na]+, and [M+H-H2O]+, were detected. In negative ion mode, [MH]- and [M+COOH]- ions were mainly analyzed. The data processing workflow included key steps such as peak extraction, retention time alignment (using the optimal QC sample as a reference), peak intensity normalization, compound deconvolution, and structure identification. For quality control, local polynomial regression was used to correct the sample signals, and ion characteristics with a relative standard deviation greater than 30% in the QC samples were removed. All analytical steps used the software's default optimized parameters to ensure data reliability. Qualitative analysis was performed by comparison with the NIST 20 standard spectral library, and semi-quantitative analysis was performed using the ratio of internal standard compound concentration to volatile compound concentration.

[0086] like Figure 9 As shown, a total of 59 volatile components were identified in the three groups of samples, covering nine major categories of compounds: sulfur-containing compounds (1 type), phenols (2 types), nitrogen-containing compounds (5 types), ketones (15 types), furans (6 types), esters (13 types), aldehydes (5 types), alcohols (8 types), and acids (4 types). Compared with the blank group (Group A) and the control group (Group D), the total amount of volatile compounds in the experimental group (Group E) of high-temperature Daqu increased from 96.24 ng / g and 496.80 ng / g to 993.33 ng / g, respectively, representing increases of approximately 9 times and 1 times.

[0087] Many volatile compounds across various classes of compounds showed significant increases in volatile content. Specifically, acetic acid, 2-methylpropionic acid, hexanoic acid, n-hexanol, 2-ethyl-1-hexanol, methanol, nonanal, phenylacetaldehyde, hexanal, 3-methylbutanal, 3,7-dimethyl-6-octenyl decanoate, decyl-1-propenyl-2-yl carbonate, 3-hydroxy-2,2,4-trimethylpentyl 2-methylpropionic acid, methyl acetate, methyl hexanoate, ethyl acetate, 3-methylfuran, 3-furanaldehyde, 2-furanethanol, 2-furanaldehyde, 3-methyl-2-butanone, 2-hexanone, 4,6-dimethyl-2-heptanone, 2-butanone, methyl isobutyl ketone, acetone, 2-pentanone, tetramethylpyrazine, 2,4-di-tert-butylphenol, and dimethyl disulfide all showed significant increases. Figure 10 Among the experimental results, the contents of 3-methylfuran, 3-furanaldehyde, 2-furanethanol, and 2-furanaldehyde (furfural) showed a significant increase. Furfural and 3-methylfuran together contribute the "roasted aroma" to the high-temperature Daqu (a type of fermented koji), while the fruity sweetness of 2-furanethanol neutralizes the bitterness of furfural, enhancing the flavor complexity of Daqu. The significant increase in the total nitrogen content is mainly reflected in tetramethylpyrazine, which accounts for 99.3% of the total nitrogen content in the experimental group, contributing the main "caramelized aroma" and "nutty aroma" to Daqu. It can form a "caramelized aroma-nutty aroma" complex with furan compounds such as furfural. The presence of trace sulfur compounds—dimethyl disulfide—enhances the penetrating power of the soy sauce aroma. The addition of bacteriophages altered the microbial community structure during Daqu fermentation, producing more unique metabolites.

[0088] In summary, the bacteriophage provided by this invention can be added to the raw materials for Daqu production, enabling Daqu to produce more characteristic compounds, giving Daqu a more layered flavor and a richer "qu aroma", and has good research and development value and application prospects in the field of Baijiu brewing.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0090] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0091] The fact that the steps of the method are listed in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A lytic Lactobacillus plantarum phage strain ZMDQ02, with accession number CGMCC NO.46160.

2. A kit comprising bacteriophage ZMDQ02 and its preservation solution, wherein the preservation number of bacteriophage ZMDQ02 is CGMCC NO.46160.

3. A phage preparation comprising phage ZMDQ02 and Lactobacillus plantarum, wherein the preservation number of phage ZMDQ02 is CGMCC NO.46160.

4. The application of bacteriophage ZMDQ02, or the bacteriophage preparation according to claim 3, in the production of Daqu (a type of starter culture) and Baijiu (Chinese liquor); The preservation number of the bacteriophage ZMDQ02 is CGMCC NO.46160; Preferably, the Daqu (a type of starter culture) is high-temperature Daqu or medium-high-temperature Daqu.

5. A method for producing Daqu (a type of starter culture), comprising the steps of adding bacteriophage ZMDQ02 or the bacteriophage preparation of claim 3 to pulverized Daqu raw materials and then culturing them; The preservation number of the bacteriophage ZMDQ02 is CGMCC NO.46160; Preferably, the raw material for the Daqu (a type of starter culture) is selected from one or more of wheat, barley, and peas, especially wheat; Preferably, the method further includes the step of pressing the resulting raw material mixture into koji blocks.

6. The method as described in claim 5, characterized in that, The method includes the following steps: (1) Moistening wheat: Adding water to wheat; (2) Grinding: Grind the wheat obtained in step (1); (3) Mixing and pressing: Add water, mother koji, bacteriophage ZMDQ02 or the bacteriophage preparation described in claim 3 to the pulverized raw material obtained in step (2), put it into a mold, and press it into shape; (4) Cultivation: Cultivate bacteria in the slurry obtained in step (3).

7. The method as described in claim 6, characterized in that, In step (1), the proportion of water added is 1%-10% of the wheat, v / w; Preferably, the soaking time in step (1) is 8-24 hours.

8. The method as described in claim 6, characterized in that, In step (3), the proportion of water added is 35%-45% of the wheat, v / w; Preferably, the proportion of mother koji added in step (3) is 5%-20% of wheat, w / w.

9. The method according to any one of claims 6-8, characterized in that, In step (3), the inoculation amount of Lactobacillus plantarum is 0.5%-5%, v / w; and / or, in step (3), the amount of phage added is based on a multiplicity of infection of 1-5; Preferably, in step (3), the inoculation amount of Lactobacillus plantarum is 1%, and the multiplicity of infection of the bacteriophage is 1.

10. The Daqu produced by the method according to any one of claims 5-9.

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