A lactobacillus plantarum with no production of biogenic amine and broad spectrum degradation capacity, its selection method and application

Abstract

The application provides a plant lactobacillus with no production of biogenic amine and wide spectrum degradation capacity, a selection method and application thereof, and relates to the technical field of microorganisms. The selection method of the plant lactobacillus H2-4 comprises the following steps: taking the plant lactobacillus NCUTUAS4 as a starting strain, performing ARTP mutagenesis treatment, and taking no production of biogenic amine and wide spectrum degradation capacity as screening indexes to obtain the mutant plant lactobacillus H2-4. The mutant strain has wide spectrum degradation capacity on seven common biogenic amines, has strong acid production capacity, good alcohol tolerance and high genetic stability, and can be widely applied in fermented wine such as rice wine and yellow rice wine, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a plant lactobacillus that does not produce bioamines and has a broad-spectrum degradation ability, its breeding method and application. Background Technology

[0002] Rice wine is a traditional Chinese brewed alcoholic beverage. Its brewing process involves semi-open fermentation, resulting in a complex microbial environment that easily accumulates potentially harmful substances—biogenic amines. Biogenic amines are a class of low-molecular-weight nitrogen-containing basic organic compounds, mainly including tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. Excessive intake of biogenic amines can cause adverse reactions such as headaches, nausea, palpitations, and abnormal blood pressure, and in severe cases, can endanger life. Studies have shown that the biogenic amine content in rice wine is much higher than in other alcoholic beverages such as beer and wine, reaching 3–260 mg / L, posing a significant food safety risk.

[0003] Lactic acid bacteria are one of the dominant microbial groups in the fermentation process of rice wine, and are also an important source of biogenic amines, especially Lactobacillus and Enterococcus. How to effectively control the generation of biogenic amines during the fermentation process of rice wine is a technical problem that urgently needs to be solved in this field. At present, the technical strategies for controlling the content of biogenic amines in fermented foods in the existing technology mainly include the following categories: (1) Optimize the fermentation production process: inhibit the growth and metabolism of amine-producing microorganisms by adjusting the process conditions such as temperature, pH value, salt concentration, and fermentation time. Although this method can reduce the generation of biogenic amines to a certain extent, it cannot degrade the biogenic amines that have already been generated, and excessive adjustment of process conditions may affect the flavor quality of the product; (2) Reduce the number of amine-producing microorganisms: obtain lactic acid bacteria strains that do not produce biogenic amines by screening or mutagenesis, and replace the original amine-producing strains in the fermentation. However, the above methods have the defect of "functional separation": optimizing the process or screening non-amine-producing strains can only passively inhibit the generation of biogenic amines, but cannot actively remove the existing biogenic amines brought in by the raw materials or coexisting microorganisms. In actual fermentation systems, the raw materials themselves or coexisting microorganisms may still introduce biogenic amines. Simply using non-amine-producing strains cannot solve the problem of the accumulation of biogenic amines that have already been generated; (3) Adding inhibitors to suppress the metabolic activity of amine-producing microorganisms: However, such methods have poor selectivity and may simultaneously adsorb or destroy flavor substances and nutrients in the wine, and there are safety controversies. Moreover, none of these methods can effectively degrade the biogenic amines that have already been generated.

[0004] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0005] The purpose of this invention is to provide a plant lactobacillus that does not produce bioamines and has a broad-spectrum degradation ability, its breeding method and application.

[0006] In a first aspect, the present invention provides a plant lactobacillus that does not produce bioamines and has broad-spectrum degradation capabilities. The plant lactobacillus is plant lactobacillus H2-4, which was deposited on May 25, 2026 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261085, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] Optionally, the *Lactobacillus plantarum* H2-4 does not produce biological amines and has the ability to degrade tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. In MRS liquid medium containing tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine at an initial concentration of 100 mg / L, after culturing at 37°C for 120 h, the degradation rate of spermine is ≥85%, the degradation rate of spermidine is ≥70%, and the degradation rate of tyramine is ≥60%.

[0008] Optionally, the total acid content of the *Lactobacillus plantarum* H2-4 after being cultured in MRS liquid medium at 37°C for 48 hours is ≥10 g / L; the relative growth rate in a medium containing 8% ethanol is ≥15%; and it is genetically stable after 10 consecutive passages.

[0009] Secondly, the present invention provides a method for breeding any of the above-mentioned optional Lactobacillus plantarum, comprising the following steps:

[0010] (1) Using Lactobacillus plantarum NCUTUUAS4 as the starting strain, a bacterial suspension was prepared after activation culture to the logarithmic growth phase;

[0011] (2) The bacterial suspension prepared in step (1) was subjected to mutagenesis using atmospheric pressure room temperature plasma;

[0012] (3) Spread the bacterial culture after mutagenesis in step (2) onto MRS solid medium, pick a single colony and inoculate it into amino acid decarboxylase liquid medium, and screen for mutant strains that do not produce bioamines.

[0013] (4) The mutant strains that do not produce bioamines obtained in step (3) are inoculated into MRS liquid medium containing bioamines, the bioamine degradation rate is measured, and mutant strains with bioamine degradation ability are obtained by re-screening.

[0014] (5) The fermentation performance of the mutant strains with biogenic amine degradation ability obtained by secondary screening was evaluated to obtain the target strain Lactobacillus plantarum H2-4.

[0015] Optionally, the activation culture in step (1) is as follows: *Lactobacillus plantarum* NCUTUUAS4 is inoculated into MRS liquid medium and statically cultured at 35-37℃ for 8-12 hours to bring it into the logarithmic growth phase; the concentration of the bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0016] Optionally, the parameters of the mutagenesis treatment in step (2) are: working gas is helium, gas flow rate is 10L / min, power is 120W, treatment distance is 2mm, ambient temperature is 20℃; mutagenesis time is 75-85s, and the mutagenesis time makes the lethality rate 85%-95%.

[0017] Optionally, when picking a single colony and inoculating it into an amino acid decarboxylase liquid culture medium as described in step (3), the biogenic amine content is determined after culturing at 35-37℃ for 3-5 days.

[0018] Optionally, the biogenic amines mentioned in step (4) include tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0019] Optionally, the fermentation performance evaluation in step (5) includes evaluation of acid production capacity, evaluation of alcohol tolerance and evaluation of genetic stability.

[0020] Thirdly, the present invention provides the application of any of the above-mentioned optional *Lactobacillus plantarum* or *Lactobacillus plantarum* obtained by any of the above-mentioned optional methods in fermented foods, including inoculating the *Lactobacillus plantarum* and yeast into a fermentation system in a time sequence for mixed fermentation, in order to reduce the content of biogenic amines in fermented foods.

[0021] Optionally, the fermented food includes one of rice wine, yellow wine, wine, and beer.

[0022] Optionally, the yeast includes aroma-producing yeast and brewer's yeast; the timing sequence is to first inoculate the *Lactobacillus plantarum* and the aroma-producing yeast, and then inoculate the brewer's yeast; the brewer's yeast is added in the form of pure culture liquid or in the form of rice wine koji containing brewer's yeast.

[0023] Optionally, the *Lactobacillus plantarum*, the aroma-producing yeast, and the rice wine starter containing *Saccharomyces cerevisiae* are subjected to mixed fermentation according to the following steps:

[0024] (1) The glutinous rice raw material is steamed and saccharified to obtain fermented mash;

[0025] (2) On day 0, the plant lactobacillus and the aroma-producing yeast were inoculated into the fermented mash; the aroma-producing yeast was Wickham yeast.

[0026] (3) After culturing for 20-28 hours, inoculate with the rice wine starter containing brewing yeast;

[0027] (4) Primary fermentation and secondary fermentation are carried out to obtain rice wine.

[0028] Optionally, the inoculum size of *Lactobacillus plantarum* in step (2) is 1 × 10⁻⁶. 5 -1×106 CFU / mL, the inoculum size of the abnormal Wickham yeast was 1×10⁻⁶. 7 -2×10 7 CFU / mL; the inoculation amount of the rice wine starter containing brewing yeast in step (3) is 0.6%-2%.

[0029] Optionally, the primary fermentation temperature is 28°C and the primary fermentation time is 5 days; the secondary fermentation temperature is 15°C and the secondary fermentation time is 15 days.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The breeding method provided by the present invention uses Lactobacillus plantarum NCUTUUAS4 as the starting strain and adopts ambient pressure room temperature plasma (ARTP) mutagenesis treatment. It uses the absence of bioamine production and the ability to degrade a wide range of substances as the dual screening indicators. The operation is safe and the mutation efficiency is high, which can quickly obtain functionally integrated mutant strains.

[0032] (2) The plant lactobacillus H2-4 obtained in this invention does not produce any biogenic amines and has the ability to degrade seven biogenic amines, including tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine. The degradation rates of spermine and spermidine are as high as 90.46% and 77.53%, respectively, overcoming the technical defects of existing strains with narrow degradation spectrum and poor ability to degrade long-chain biogenic amines.

[0033] (3) The plant lactobacillus H2-4 obtained by this invention has strong acid production capacity (the total acid content reaches 12.06 g / L after 48 h of culture at 37℃ in MRS liquid medium), good alcohol tolerance (the relative growth rate reaches 19.49% in an environment containing 8% ethanol), and genetic stability (it basically does not produce biological amines after 10 consecutive generations), making it suitable for application in actual fermentation systems.

[0034] (4) In this invention, Lactobacillus plantarum H2-4 is mixed with Wickham yeast and Saccharomyces cerevisiae according to the WL-S inoculation strategy (Lactobacillus plantarum H2-4 and Wickham yeast are inoculated first, and Saccharomyces cerevisiae is inoculated 1 day later). This timing regulation strategy makes full use of the early colonization advantage of aroma-producing yeast and lactic acid bacteria, creating a favorable microenvironment for subsequent alcoholic fermentation, achieving a synergistic improvement of "amine reduction" and "aroma enhancement", and significantly improving the flavor of the product while reducing the food safety risk of fermentation.

[0035] (5) The strains and fermentation process provided by this invention can be widely applied to the production of traditional fermented wines such as rice wine and yellow wine, and have significant economic and social benefits. Attached Figure Description

[0036] Figure 1 The ARTP-induced mutagenic mortality curve;

[0037] Figure 2 Bar chart showing the degradation rates of seven biogenic amines by the mutant strain *Lactobacillus plantarum* H2-4;

[0038] Figure 3 The acid-producing capacity of the five non-bioamine-producing strains (numbered 2-4, 3-1, 3-2, 3-8, and 4-2) obtained in step (3) of Example 1 of this invention;

[0039] Figure 4 Alcohol tolerance of the five non-bioamine-producing strains (numbered 2-4, 3-1, 3-2, 3-8, and 4-2) obtained in step (3) of Example 1 of this invention;

[0040] Figure 5 To assess the genetic stability of the mutant strain *Lactobacillus plantarum* H2-4;

[0041] Figure 6 This is a graph showing the effect of different inoculation sequences on the total biogenic amine content in mixed-culture fermentation in Example 2 of the present invention.

[0042] Figure 7 This is a comparison of the total biogenic amine content of rice wine in the experimental group (CG2) and the control group (CG1) of Example 3 of the present invention;

[0043] Figure 8 This is a comparison of the content of volatile flavor compounds in rice wine of the experimental group (CG2) and the control group (CG1) in Example 3 of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0045] This invention provides a plant lactobacillus that does not produce bioamines and has broad-spectrum degradation capabilities. The plant lactobacillus is plant lactobacillus H2-4, classified and named Lactobacillus plantarum H2-4, and was deposited on May 25, 2026 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261085. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0046] Specifically, the *Lactobacillus plantarum* NCTUAS4 used, classified and named *Lactobacillus plantarum* NCTUAS4, was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 30, 2024, with accession number CCTCC NO: M 2024858. This *Lactobacillus plantarum* NCTUAS4 has been disclosed in Chinese patent literature (publication number: CN118813492A, title: A composite mutagenized *Lactobacillus plantarum* and its mutagenization method and application).

[0047] In some embodiments, *Lactobacillus plantarum* H2-4 does not produce biological amines and has the ability to degrade tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. In MRS liquid medium containing 100 mg / L of tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, after culturing at 37°C for 120 h, the degradation rate of spermine is ≥85%, the degradation rate of spermidine is ≥70%, and the degradation rate of tyramine is ≥60%.

[0048] Specifically, the mutant strain *Lactobacillus plantarum* H2-4 was inoculated into MRS liquid medium containing seven biogenic amines (tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, all at a concentration of 100 mg / L). The degradation rates of each biogenic amine were determined by pre-column derivatization using HPLC. The degradation rates of tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine were 44.08%, 36.42%, 15.66%, 4.18%, 67.69%, 77.53%, and 90.46%, respectively. In particular, the degradation rates of spermine and spermidine were as high as 90.46% and 77.53%, respectively.

[0049] In some embodiments, *Lactobacillus plantarum* H2-4, after being cultured in MRS liquid medium at 37°C for 48 hours, has a total acid content ≥10 g / L, a relative growth rate ≥15% in a medium containing 8% ethanol, and is genetically stable after 10 consecutive passages.

[0050] Specifically, the mutant strain *Lactobacillus plantarum* H2-4, when cultured in MRS liquid medium at 37°C for 48 hours, had a total acid content of 12.06 g / L; a relative growth rate of 19.49% in an 8% ethanol environment; and was genetically stable after 10 consecutive passages.

[0051] The present invention also provides a method for breeding the above-mentioned *Lactobacillus plantarum* H2-4, comprising the following steps:

[0052] (1) Using Lactobacillus plantarum NCUTUUAS4 as the starting strain, a bacterial suspension was prepared after activation culture to the logarithmic growth phase;

[0053] (2) The bacterial suspension prepared in step (1) was subjected to mutagenesis using atmospheric pressure room temperature plasma;

[0054] (3) Spread the bacterial culture after mutagenesis in step (2) onto MRS solid medium, pick a single colony and inoculate it into amino acid decarboxylase liquid medium, and screen for mutant strains that do not produce bioamines.

[0055] (4) The mutant strains that do not produce bioamines obtained in step (3) are inoculated into MRS liquid medium containing bioamines, the bioamine degradation rate is measured, and mutant strains with bioamine degradation ability are obtained by re-screening.

[0056] (5) The fermentation performance of the mutant strains with biogenic amine degradation ability obtained by secondary screening was evaluated to obtain the target strain Lactobacillus plantarum H2-4.

[0057] In some embodiments, the activation culture in step (1) is as follows: *Lactobacillus plantarum* NCUTUUAS4 is inoculated into MRS liquid medium and statically cultured at 35-37℃ for 8-12 hours to bring it into the logarithmic growth phase; the concentration of the obtained bacterial suspension is 1×10⁻⁶. 8 CFU / mL.

[0058] Specifically, the preparation of the bacterial suspension includes taking 5 mL of *Lactobacillus plantarum* NCUTUUAS4 inoculated in the logarithmic growth phase, centrifuging to collect the bacterial cells, washing three times with PBS buffer, resuspending, and adjusting the concentration to 1×10⁻⁶. 8 CFU / mL.

[0059] In some embodiments, the parameters of the mutagenesis treatment used in step (2) are: working gas is helium, gas flow rate is 10L / min, power is 120W, treatment distance is 2mm, ambient temperature is 20℃; mutagenesis time is 75-85s, and the mutagenesis time makes the lethality rate 85%-95%.

[0060] Specifically, the bacterial suspension was mutagenized using ambient pressure room temperature plasma (ARTP) for 80 seconds, resulting in a mortality rate of 89.02%.

[0061] In some embodiments, when picking a single colony in step (3) and inoculating it into an amino acid decarboxylase liquid culture medium, the biogenic amine content is measured after culturing at 35-37℃ for 3-5 days, and the absence of any biogenic amine is considered a positive initial screening result.

[0062] In some embodiments, the biogenic amines in step (4) include tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine.

[0063] Specifically, the mutant strains that do not produce bioamines obtained in step (3) were inoculated into MRS liquid culture medium containing seven bioamines (tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine, each bioamine having a concentration of 100 mg / L). The degradation rate of each bioamine was determined by pre-column derivatization using HPLC, and mutant strains that efficiently degrade bioamines were obtained through rescreening.

[0064] In some embodiments, the fermentation performance evaluation in step (5) includes evaluation of acid production capacity, alcohol tolerance, and genetic stability.

[0065] Specifically, the acid production capacity was evaluated by inoculating the selected mutant strain *Lactobacillus plantarum* H2-4 into MRS liquid medium, culturing at 37°C for 48 h, and then measuring the total acid content. Alcohol tolerance was evaluated by culturing in MRS medium containing different ethanol concentrations at 37°C for 24 h, and then measuring the OD value. 600 The relative growth rate was calculated; the genetic stability was evaluated by subculturing the strains 10 times consecutively and determining their non-producible amine properties.

[0066] In fact, the mutant strain *Lactobacillus plantarum* H2-4, after screening, showed a total acid content of 12.06 g / L when cultured in MRS liquid medium at 37°C for 48 h; a relative growth rate of 19.49% in an 8% ethanol environment; and genetic stability after 10 consecutive passages.

[0067] The present invention also provides an application of the above-mentioned *Lactobacillus plantarum* H2-4 or the above-mentioned *Lactobacillus plantarum* H2-4 obtained by any of the above optional methods in fermented foods, including inoculating the *Lactobacillus plantarum* and yeast into the fermentation system in a time sequence for mixed fermentation, in order to reduce the content of biogenic amines in fermented foods.

[0068] In some embodiments, the fermented food prepared includes one of rice wine, yellow wine, wine, and beer.

[0069] In some embodiments, the yeasts used include aroma-producing yeast and brewer's yeast; the sequence of inoculation is to first inoculate *Lactobacillus plantarum* H2-4 and aroma-producing yeast, and then inoculate brewer's yeast; the brewer's yeast is added in the form of pure culture liquid or in the form of rice wine koji containing brewer's yeast.

[0070] In some embodiments, *Lactobacillus plantarum* H2-4, aroma-producing yeast, and rice wine starter containing *Saccharomyces cerevisiae* are mixed-culture fermented according to the following steps:

[0071] (1) The glutinous rice raw material is steamed and saccharified to obtain fermented mash;

[0072] (2) On day 0, the plant lactobacillus and the aroma-producing yeast were inoculated into the fermented mash; the aroma-producing yeast was Wickham yeast.

[0073] (3) After culturing for 20-28 hours, inoculate with the rice wine starter containing brewing yeast;

[0074] (4) Primary fermentation and secondary fermentation are carried out to obtain rice wine.

[0075] Specifically, the process of obtaining fermented glutinous rice in step (1) includes: steaming glutinous rice and cooling it, then adding Angel saccharification starter (4 grams of saccharification starter per kilogram of dry glutinous rice), mixing well, and then saccharifying in a 28°C incubator for 24 hours to obtain fermented glutinous rice.

[0076] In some embodiments, the inoculum size of *Lactobacillus plantarum* H2-4 in step (2) is 1 × 10⁻⁶. 5 -1×10 6 CFU / mL, the inoculum size of *Wickhamia lanceolata* was 1×10⁻⁶. 7 -2×10 7 CFU / mL; the inoculation amount of rice wine starter containing brewing yeast in step (3) is 0.6%-2%.

[0077] Specifically, in step (2), the preferred inoculation amounts of the mutant strain *Lactobacillus plantarum* H2-4 and the abnormal *Wickham's yeast* are 2.4 × 10⁻⁶. 5 CFU / mL and 1×10 7 CFU / mL.

[0078] Specifically, in step (3), it is preferable to inoculate with rice wine starter containing brewing yeast after culturing for 24 hours, and the inoculation amount is preferably 1%.

[0079] In some embodiments, the primary fermentation temperature is 28°C and the primary fermentation time is 5 days; the secondary fermentation temperature is 15°C and the secondary fermentation time is 15 days.

[0080] In fact, when the mutant strain *Lactobacillus plantarum* H2-4 was mixed with *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae* containing rice wine yeast for fermentation according to the above-mentioned WL-S strategy, compared with the control without inoculation of mutant strain *Lactobacillus plantarum* H2-4 and *Saccharomyces cerevisiae*, the total biogenic amine content in the resulting rice wine was reduced by more than 36.6%, the total ester content was increased by more than 19.21%, and the sensory score was increased by more than 10 points.

[0081] The strains and reagents used in the following examples are from the following sources:

[0082] 1. Aberrant Wickhamomyces NCUF 307.1: The aberrant Wickhamomyces NCUF 307.1 used in Example 2 of this invention is classified as Wickerhamomyces anomalus NCUF 307.1. It was deposited at the China Center for Type Culture Collection on September 26, 2021, with accession number CCTCC NO: M 20211219. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0083] 2. Saccharomyces cerevisiae Y10: The Saccharomyces cerevisiae Y10 used in this invention has been disclosed in a master's thesis (see: Zeng Jiali. Effects of Lactobacillus plantarum on the flavor and quality of navel orange wine fermented with Saccharomyces cerevisiae and Wickham's aberrant yeast [D]. Nanchang University, 2024. DOI:10.27232 / d.cnki.gnchu.2024.004853.). The preparation process of the Saccharomyces cerevisiae Y10 used is as follows:

[0084] I. Strains Isolation and Purification

[0085] Single yeast colonies were isolated and purified from the medicinal wine sample. The test strain was streaked onto WLN agar plates and incubated at 28°C for 48 hours. Single colonies were selected for observation of morphology, color, size, edge regularity, and surface protrusion. The test strain was then streaked onto YPD agar plates and incubated at 28°C for 48 hours. Single colonies were then inoculated into YPD liquid medium and incubated at 28°C and 180 rpm for 24 hours. Cell morphology was then observed under a microscope.

[0086] II. Morphological Classification of Yeasts

[0087] Taking advantage of the characteristic that *Saccharomyces cerevisiae* cannot grow using lysine as its sole nitrogen source, the tested strains were streaked in LYS medium, and their growth was observed. Strains that did not grow normally were preliminarily identified as *Saccharomyces cerevisiae*, while those that grew normally were classified as aroma-producing yeasts. Based on this method, the target strain preliminarily identified as *Saccharomyces cerevisiae* was named *Saccharomyces cerevisiae* Y10.

[0088] III. Screening of brewing yeasts with high ethanol and low methanol production

[0089] (a) Initial screening of TTC plates (qualitative screening of alcohol production capacity)

[0090] The activated strain was diluted to an appropriate gradient, and 10 μL was spotted onto the lower layer of TTC agar plates and incubated at 28°C for 1-2 days. After colonies grew, the upper layer of TTC agar was poured in under aseptic conditions to completely cover the original colonies, and the plates were incubated at 28°C in the dark for 6-12 hours. TTC (2,3,5-triphenyltetrazolium chloride) served as a chromogenic agent, reacting with reducing substances produced by microbial metabolism to generate a red substance. The darker the color of the agar plate, the stronger the ethanol-producing ability of the strain. Based on this, strains with strong ethanol-producing ability were screened.

[0091] (ii) Dunaliella salina rescreening (rescreening of fermentation ability)

[0092] The strains obtained from the initial screening were activated in 10 mL of YPD liquid medium at 28°C for 18-24 h, and then 1×10⁻⁶ were added. 6 Inoculation was performed at a concentration of CFU / mL in YPD liquid medium containing Durham tubes, and the culture was incubated statically at 28°C. Fermentation was observed by monitoring the trapped air bubbles within the Durham tubes. Strains that filled the Durham tubes with air bubbles within 48 hours were considered to have good fermentation ability, while other strains were considered to have poor fermentation ability. Based on this, strains with excellent fermentation ability were selected.

[0093] (III) Determination of alcohol production capacity

[0094] Following the Perez-Martin method, the seed culture of the selected superior strains was diluted with 1×10⁻⁶. 6 The inoculum was inoculated into a fermentation medium with a sugar content of 23°Bx at a CFU / mL, and fermented statically at 28°C for 7 days. The ethanol content was determined using the potassium dichromate method.

[0095] (iv) Determination of methanol production capacity

[0096] After juicing the navel oranges, the sugar content was adjusted to 23°Bx. The seed liquids of each strain were then diluted with 1×10⁻⁶ molasses. 6 The inoculum was inoculated into navel orange juice medium at a concentration of CFU / mL and fermented statically at 20°C for 7 days. The methanol content in the fermentation broth was determined by gas chromatography according to GB5009.266—2016.

[0097] The chromatographic conditions were as follows: The column was a DB-FATWAX UI (30m × 250μm × 0.25μm); the column temperature program was: initial temperature 40℃, held for 1 min, increased to 130℃ at 4.0℃ / min, then increased to 200℃ at 20℃ / min, held for 5 min; detector temperature 250℃; injection port temperature 250℃; carrier gas flow rate 1.0 mL / min; injection volume 1.0 μL; split ratio 20:1. The methanol content was calculated using the following formula: X = ρ, where X is the methanol content in the sample (mg / L), and ρ is the methanol concentration in the sample solution obtained from the standard curve (mg / L). The calculation result was retained to three significant figures.

[0098] (v) Establishment of the target strain

[0099] Based on the combined results of ethanol yield and methanol content measurements, the target strain of Saccharomyces cerevisiae Y10, which produces high ethanol and low methanol, was screened out and classified as Saccharomyces cerevisiae Y10.

[0100] 3. Angel Yeast Saccharification Starter: Purchased from Angel Yeast Co., Ltd., product batch number M46.

[0101] Example 1: ARTP mutagenesis to breed Lactobacillus plantarum H2-4, which does not produce bioamines and has a broad-spectrum bioamine degradation ability.

[0102] S1. Activation of the starting strain and preparation of bacterial suspension

[0103] Laboratory-preserved *Lactobacillus plantarum* NCUTUUAS4 (CCTCC NO: M 2024858) was inoculated into MRS liquid medium and activated twice. Then, it was transferred to fresh MRS liquid medium at a 2% inoculum size and incubated at 37°C for 8 hours (logarithmic growth phase). 5 mL of the bacterial suspension was centrifuged, the supernatant was discarded, and the suspension was washed three times with PBS buffer. The suspension was then resuspended in PBS and the concentration adjusted to 1×10⁻⁶. 8 CFU / mL (OD) 600 (≈0.8), to obtain bacterial suspension.

[0104] S2, ARTP mutagenesis treatment

[0105] Take 1 mL of the above bacterial suspension, add an equal volume of 10% glycerol as a protectant, and vortex for 2 min. Accurately pipette 10 μL of the mixture and spread it evenly onto a sterile metal slide. ARTP mutagenesis parameters: working gas is helium, flow rate is 10 L / min, power is 120 W, treatment distance is 2 mm, and ambient temperature is 20℃. Treatment time is 80 s (lethality rate 89.02%). After mutagenesis, place the slide in sterile PBS and vortex for 1 min. After appropriate dilution, spread it onto MRS solid medium and incubate at 37℃ for 2 days.

[0106] S3. Initial screening of mutants that do not produce bioamines.

[0107] Single colonies with good growth were randomly selected and inoculated into amino acid decarboxylase liquid medium. After static incubation at 37℃ for 5 days, the supernatant was collected and the content of biogenic amines was determined by HPLC. Using the original strain as a control, mutant strains that did not produce any biogenic amines were screened, and a total of 5 strains were obtained (numbered 2-4, 3-1, 3-2, 3-8, and 4-2).

[0108] S4, secondary screening of biogenic amine degradation capacity

[0109] After activation, the initially screened strains were inoculated at a 2% inoculum into MRS liquid medium (pH 5.5) containing seven biogenic amines (100 mg / L each) and incubated at 37°C for 120 h. The biogenic amine content was measured, and the degradation rate was calculated. The results are shown in Table 1.

[0110] Table 1: Degradation rates (%) of the seven biogenic amines by the five mutant strains (numbered 2-4, 3-1, 3-2, 3-8, and 4-2) obtained in step S3.

[0111]

[0112] Table 1 shows that strain 2-4 exhibits high degradation rates for tryptamine, putrescine, cadaverine, tyramine, spermidine, and spermine, demonstrating the best overall performance. It was named *Lactobacillus plantarum* H2-4. A bar chart was plotted showing the degradation rates of the seven biogenic amines by the mutant strain *Lactobacillus plantarum* H2-4. The results are as follows: Figure 2 As shown.

[0113] S5. Evaluation of acid production capacity, alcohol tolerance, and genetic stability.

[0114] Acid production capacity was evaluated as follows: Five mutant strains (numbered 2-4, 3-1, 3-2, 3-8, and 4-2) obtained in step S3 were inoculated into MRS liquid medium and cultured at 37°C for 48 h, after which the total acid content was measured. Alcohol tolerance was evaluated as follows: Five mutant strains (numbered 2-4, 3-1, 3-2, 3-8, and 4-2) were cultured at 37°C for 24 h in MRS medium containing different ethanol concentrations, after which the OD was measured. 600 The relative growth rate was calculated; genetic stability was evaluated by subculturing the strain 10 times consecutively and determining the non-amine production performance of each generation. The results are as follows: Figures 3 to 5 As shown.

[0115] Acid production capacity: When Lactobacillus plantarum H2-4 was inoculated into MRS liquid medium and cultured at 37℃ for 48 h, the total acid content was measured to be 12.06 g / L.

[0116] Alcohol tolerance: After culturing Lactobacillus plantarum H2-4 at 37°C for 24 hours in MRS medium containing 8% ethanol, the relative growth rate was 19.49%.

[0117] Genetic stability: The ability of Lactobacillus plantarum H2-4 to produce no amines was measured after 10 consecutive passages. The results showed that almost no amines were produced in each generation, indicating good genetic stability.

[0118] Example 2: Optimization of inoculation sequence for mixed-culture fermentation

[0119] S1. Mixed-culture fermentation medium (preparation of glutinous rice saccharification liquid)

[0120] Sift glutinous rice flour through a 60-mesh sieve. Take 300g of the mixture and add it to a water ratio of 1:2.5. Add 2g of liquefying enzyme, mix thoroughly, and then liquefy in a 75℃ water bath for 1 hour. Next, add 1.5g of saccharifying enzyme, mix thoroughly, and then saccharify in a 65℃ water bath for 3 hours. Filter through cheesecloth.

[0121] Incubate overnight at 4°C, and collect the supernatant to obtain the fermentation medium.

[0122] S2. Inoculation sequence for mixed fermentation (Lactobacillus plantarum H2-4 will be abbreviated as L.plantarum H2-4; Wickerhamomyces anomalus NCUF 307.1 will be classified as Wickerhamomyces anomalus NCUF 307.1, abbreviated as W. anomalus 307.1; Saccharomyces cerevisiae Y10 will be classified as Saccharomyces cerevisiae Y10, abbreviated as S. cerevisiae Y10.)

[0123] Group S: S. cerevisiae Y10 pure fermentation

[0124] Group W: W. anomalus 307.1 pure fermentation

[0125] Group SW: S. cerevisiae Y10 and W. anomalus 307.1 were simultaneously inoculated for fermentation.

[0126] WS1 group: Inoculated with *W. anomalus* 307.1 first, followed by *S. cerevisiae* Y10 24 hours later.

[0127] WS2 group: Inoculated with *W. anomalus* 307.1 first, followed by *S. cerevisiae* Y10 48 hours later.

[0128] WL-S group: W. anomalus 307.1 and L. plantarum H2-4 were administered first, followed by S. cerevisiae Y10 24 hours later.

[0129] W-LS group: W. anomalus 307.1 was administered first, followed by simultaneous administration of L. plantarum H2-4 and S. cerevisiae Y10 24 hours later.

[0130] After fermentation, the biogenic amine content in group WS1 (4.41 mg / L) was lower than that in group SW (5.71 mg / L) and group WS2 (6.91 mg / L), and the biogenic amine content in group WL-S (2.15 mg / L) was lower than that in group W-LS (3.37 mg / L). The effects of different inoculation sequences on the total biogenic amine content of mixed fermentation are as follows: Figure 6 As shown.

[0131] Example 3: Application of Lactobacillus plantarum H2-4 in rice wine brewing

[0132] S1. Preparation of fermented rice wine mash

[0133] Soak glutinous rice for 24 hours, drain and steam for 15-20 minutes. After cooling, add Angel saccharification starter (4g / kg glutinous rice) and saccharify at 28℃ for 24 hours to obtain fermented mash.

[0134] S2, Mixed-culture fermentation strategy

[0135] Experimental group (CG2): *Wickham's abnormal yeast* NCUF 307.1 and *Lactobacillus plantarum* H2-4 were first inoculated into the fermented mash at inoculation amounts of 1×10⁻⁶. 7 CFU / mL and 2.4×10 5 CFU / mL; after 24 hours, add 1% rice wine starter (containing brewing yeast); primary fermentation at 28℃ for 5 days, followed by secondary fermentation at 15℃ for 15 days.

[0136] Control group (CG1): After saccharification, only 1% rice wine koji (containing brewing yeast) was inoculated, and other conditions were the same as those of the experimental group (CG2).

[0137] S3, Result Determination

[0138] After fermentation, the biogenic amine content, volatile flavor compounds, and sensory scores of the rice wine in the experimental group (CG2) and the control group (CG1) were measured. The results are as follows: Figures 7 to 8 As shown in Table 2.

[0139] Biogenic amines: The total biogenic amine content in group CG2 was 10.10 mg / L, which was 36.6% lower than that in group CG1 (15.93 mg / L).

[0140] Volatile flavor compounds: The total volatile flavor compound content of group CG2 increased by 9.71% compared with group CG1, of which esters increased by 19.21% and alcohols increased by 7.57%.

[0141] Sensory rating: The total score of CG2 group was 84.75, which was significantly higher than that of CG1 group (74.25), indicating a rich aroma and harmonious taste.

[0142] Table 2: Sensory Evaluation Results

[0143]

[0144] The results in Table 2 above show that the *Lactobacillus plantarum* H2-4 and its mixed-culture fermentation strategy provided by this invention can effectively reduce the content of biogenic amines in rice wine and significantly improve its flavor quality.

[0145] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A plant lactobacillus that does not produce bioamines and has broad-spectrum degradation capabilities, characterized in that, The Lactobacillus plantarum mentioned is Lactobacillus plantarum H2-4, which was deposited at the China Center for Type Culture Collection on May 25, 2026, with accession number CCTCC NO: M 20261085, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A method for breeding *Lactobacillus plantarum* as described in claim 1, characterized in that, Includes the following steps: (1) Using Lactobacillus plantarum NCUTUUAS4 as the starting strain, a bacterial suspension was prepared after activation culture to the logarithmic growth phase; (2) The bacterial suspension prepared in step (1) was subjected to mutagenesis using atmospheric pressure room temperature plasma; (3) Spread the bacterial culture after mutagenesis in step (2) onto MRS solid medium, pick a single colony and inoculate it into amino acid decarboxylase liquid medium, and screen for mutant strains that do not produce bioamines. (4) The mutant strains that do not produce bioamines obtained in step (3) are inoculated into MRS liquid medium containing bioamines, the bioamine degradation rate is measured, and mutant strains with bioamine degradation ability are obtained by re-screening. (5) The fermentation performance of the mutant strains with biogenic amine degradation ability obtained by secondary screening was evaluated to obtain the target strain Lactobacillus plantarum H2-4.

3. The method according to claim 2, characterized in that, The activation culture described in step (1) is as follows: *Lactobacillus plantarum* NCUTUUAS4 is inoculated into MRS liquid medium and statically cultured at 35-37℃ for 8-12 hours to bring it into the logarithmic growth phase; the concentration of the bacterial suspension is 1×10⁻⁶. 8 CFU / mL; and / or, the parameters of the mutagenesis treatment in step (2) are: working gas is helium, gas flow rate is 10L / min, power is 120W, treatment distance is 2mm, ambient temperature is 20℃; mutagenesis time is 75-85s; and / or, when picking a single colony and inoculating it into the amino acid decarboxylase liquid medium in step (3), the biogenic amine content is determined after culturing at 35-37℃ for 3-5 days; and / or, the biogenic amines in step (4) include tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine; and / or, the fermentation performance evaluation in step (5) includes acid production capacity evaluation, alcohol tolerance evaluation and genetic stability evaluation.

4. The application of *Lactobacillus plantarum* as described in claim 1 or *Lactobacillus plantarum* selected by the method described in any one of claims 2-3 in fermented foods, characterized in that... The *Lactobacillus plantarum* and yeast were inoculated into the fermentation system in a sequential manner for mixed fermentation, which was used to reduce the content of biogenic amines in fermented foods.

5. The application according to claim 4, characterized in that, The fermented food includes one of rice wine, yellow wine, wine, and beer.

6. The application according to claim 4, characterized in that, The yeasts include aroma-producing yeast and brewer's yeast; the inoculation sequence is to first inoculate the *Lactobacillus plantarum* and the aroma-producing yeast, and then inoculate the brewer's yeast; the brewer's yeast is added in the form of pure culture liquid or in the form of rice wine koji containing brewer's yeast.

7. The application according to claim 6, characterized in that, The *Lactobacillus plantarum*, the aroma-producing yeast, and the rice wine starter containing brewing yeast are subjected to mixed fermentation according to the following steps: (1) The glutinous rice raw material is steamed and saccharified to obtain fermented mash; (2) On day 0, the *Lactobacillus plantarum* and the aroma-producing yeast were inoculated into the fermented mash; the aroma-producing yeast was *Saccharomyces cerevisiae*. (3) After culturing for 20-28 hours, inoculate with the rice wine starter containing brewing yeast; (4) Primary fermentation and secondary fermentation are carried out to obtain rice wine.

8. The application according to claim 7, characterized in that, The inoculation amount of *Lactobacillus plantarum* in step (2) is 1 × 10⁻⁶. 5 -1×10 6 CFU / mL, the inoculum size of the abnormal Wickham yeast was 1×10⁻⁶. 7 -2×10 7 CFU / mL; the inoculation amount of the rice wine starter containing brewing yeast in step (3) is 0.6%-2%.

9. The application according to claim 8, characterized in that, The primary fermentation temperature is 28℃ and the primary fermentation time is 5 days; the secondary fermentation temperature is 15℃ and the secondary fermentation time is 15 days.

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