Application of phytobacterium plantarum LWQ17 in preparation of biological preparation for inhibiting putrefying bacteria of steamed buns

Abstract

The invention discloses application of plant lactobacillus LWQ17 in preparation of a biological agent for inhibiting putrefying bacteria of steamed buns, and belongs to the technical field of microorganisms. The plant lactobacillus LWQ17 obtained through screening can effectively inhibit main putrefying bacteria, namely bacillus licheniformis and bacillus amyloliquefaciens, at the high temperature in summer of the steamed buns, and the plant lactobacillus LWQ17 can destroy the integrity of cell membranes of the bacillus and increase the permeability of the cell membranes of the bacillus. According to the method, the pretreatment conditions of the steamed buns prepared through fermentation of the plant lactobacillus LWQ17 are optimized, the steamed buns are stored for 12-24 hours under the pretreatment conditions that the temperature range is 30-50 DEG C and the humidity range is 40%-85%, and the shelf life of the steamed buns is prolonged. The invention provides the method for prolonging the shelf life of the steamed buns in the normal-temperature and / or summer high-temperature environment, the method has important significance on logistics transportation and storage of the steamed buns, a new direction is provided for a biological preparation for inhibiting bacillus of the plant lactobacillus LWQ17, and the application range of the strain is also widened.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Lactobacillus plantarum LWQ17 in the preparation of biological agents that inhibit spoilage bacteria in steamed buns. Background Technology

[0002] Bacillus is widely present in natural environments such as air, soil, and water. Its heat-resistant spores are highly resilient and easily survive and germinate during food processing, leading to food spoilage and even food safety issues. Steamed buns, a traditional staple food made primarily from flour through fermentation and steaming, are rich in nutrients and have high water activity, providing favorable conditions for microbial growth. Besides mold contamination, Bacillus contamination is also a significant reason for the deterioration of steamed bun quality and shortened shelf life during storage. Especially in the hot and humid environment of summer, Bacillus multiplies rapidly, often causing steamed buns to become stringy and sticky, a phenomenon known as "rope spoilage," which severely restricts the industrial production and market circulation of steamed buns.

[0003] Fermenting sourdough with lactic acid bacteria to prepare steamed buns has become an important biological preservation method. The principle lies in the fact that lactic acid bacteria produce various natural antibacterial substances such as bacteriocins, organic acids, and antimicrobial peptides during fermentation, which inhibit various foodborne pathogens and spoilage bacteria, thus extending the shelf life of the steamed buns. Under traditional room temperature (25℃) conditions, the spoilage of steamed buns is caused by mold. However, with global warming and the development of modern online e-commerce sales models (such as express delivery), steamed buns spend more time in high-temperature environments (piled up in train carriages, transfer stations, etc.) during the summer circulation process (usually exceeding 24 hours). Sustained high temperatures inhibit mold germination and promote the growth of heat-resistant Bacillus, causing the spoilage type to change from mold-induced decay to Bacillus-induced brittle decay. This study found that specific lactic acid bacteria inhibit Bacillus growth by disrupting the cell membrane integrity of Bacillus.

[0004] Current steamed bun preservation technologies primarily focus on inhibiting mold growth at room temperature (25℃), failing to adequately consider the limitations in preservation effectiveness caused by the inability to inhibit the striation of Bacillus bacteria in the high-temperature and high-humidity environments of modern logistics. Therefore, designing a preservation method that can simultaneously inhibit mold growth at room temperature and inhibit striation caused by Bacillus bacteria in high-temperature and humid environments, thereby achieving stable and extended shelf life for steamed buns in modern logistics scenarios, is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] Based on the above needs, the purpose of this invention is to provide the application of *Lactobacillus plantarum* LWQ17 in the preparation of biological agents that inhibit spoilage bacteria in steamed buns. Through screening of lactic acid bacteria, this invention reveals that *Lactobacillus plantarum* LWQ17 can disrupt the cell membrane integrity of Bacillus and increase the cell membrane permeability of Bacillus, effectively inhibiting the main spoilage bacteria of steamed buns under high temperature and humid conditions, namely *Bacillus licheniformis* and *Bacillus amyloliquefaciens*. Combined with high-temperature pretreatment, this further extends the shelf life of steamed buns at room temperature and / or high summer temperatures, providing a novel preservation method and offering guidance for extending the shelf life of ready-to-eat pasta products such as steamed buns.

[0006] To achieve the above objectives, the present invention is implemented through the following solution: This invention provides the application of Lactobacillus plantarum LWQ17 in the preparation of biological agents that inhibit spoilage bacteria in steamed buns.

[0007] Furthermore, the preservation number of the Lactobacillus plantarum LWQ17 is CCTCC NO: M 20231803.

[0008] Furthermore, the bacteria causing the spoilage of the steamed buns include Bacillus licheniformis and / or Bacillus amyloliquefaciens.

[0009] Furthermore, the plant lactobacillus LWQ17 can disrupt the cell membrane integrity of Bacillus and increase the cell membrane permeability of Bacillus.

[0010] Furthermore, the plant lactobacillus LWQ17 can destroy the cells of Bacillus, making their surface rough and wrinkled, their shape irregular, and causing the cells to aggregate, invading, or decompose and break down.

[0011] Furthermore, the specific methods by which *Lactobacillus plantarum* LWQ17 inhibits spoilage bacteria in steamed buns include: sealing the steamed buns prepared by fermentation with *Lactobacillus plantarum* LWQ17, and pre-treating the sealed steamed buns to extend their shelf life.

[0012] Furthermore, the steamed buns are prepared from sourdough and flour. The method for preparing the sourdough is as follows: Lactobacillus plantarum LWQ17 is activated and cultured to obtain a bacterial solution. The bacterial solution is mixed with flour to prepare sourdough. The sourdough is mixed with other ingredients and kneaded until the surface of the dough is smooth. It is then placed in a fermentation box to ferment for 1 to 2 hours. The dough is then divided into 50 g to 150 g portions, shaped into balls, proofed at room temperature for 0 to 30 minutes, and steamed for 30 minutes to obtain the steamed buns.

[0013] Furthermore, the pretreatment conditions are a temperature of 30 to 50°C, a humidity of 40% to 85%, and a pretreatment time of 12 to 24 hours.

[0014] Furthermore, the preferred conditions for pretreatment of the steamed buns are a temperature of 45°C and a humidity of 85%.

[0015] Furthermore, the steamed buns are stored at 22 to 50°C.

[0016] Furthermore, the steamed buns are stored for 5 to 10 days.

[0017] Furthermore, based on the mass of flour, the amount of *Lactobacillus plantarum* LWQ17 used in the preparation of steamed buns is 10... 7 ~ 10 9 cfu / g.

[0018] This invention also provides the application of Lactobacillus plantarum LWQ17 in inhibiting Bacillus.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes LWQ17 of *Lactobacillus plantarum* to inhibit *Bacillus licheniformis* and *Bacillus amyloliquefaciens*, effectively disrupting the cell membranes of these Bacillus cells, causing the surface of the Bacillus cells to become rough and wrinkled, irregular in shape, and resulting in cell aggregation, invading, or decomposition and breakage, thereby increasing the permeability of their cell membranes.

[0020] 2. This invention screens the main spoilage bacteria of steamed buns, namely Bacillus licheniformis and Bacillus amyloliquefaciens, through experiments. It constructs an in-situ preservation technology for steamed buns using LWQ17 sourdough and combines it with high-temperature pretreatment of steamed buns to extend the shelf life of steamed buns. The storage time of steamed buns at room temperature (22 ~ 37℃) is extended by 3 ~ 8 days, and the storage time of steamed buns at high temperature in summer (45 ~ 50℃) is extended by 2 ~ 8 days.

[0021] 3. Through experimental optimization, the present invention has determined that the optimal conditions for pretreatment of steamed buns are a temperature of 45℃ and a humidity of 85%. This pretreatment method, combined with the in-situ preservation technology of Lactobacillus plantarum LWQ17 sourdough, produces steamed buns with the best storage performance. The storage time of steamed buns at room temperature and / or high temperature in summer (22~50℃) can be extended by 8 days, providing theoretical support for the steamed bun preparation process. Attached Figure Description

[0022] Figure 1 The morphology of bacteria that cause spoilage of steamed buns; Figure 2 Phylogenetic tree of strains Y-8 and Y-9; Figure 3 The images show the morphological structures of Bacillus licheniformis and Bacillus amyloliquefaciens under scanning electron microscopy. A and C represent the control groups for Bacillus licheniformis and Bacillus amyloliquefaciens, respectively, while B and D represent the LWQ17 CFS treatment groups for Bacillus licheniformis and Bacillus amyloliquefaciens, respectively. Figure 4 The effect of Lactobacillus plantarum LWQ17 CFS on the leakage of Bacillus nucleic acid, where A is Bacillus licheniformis and B is Bacillus amyloliquefaciens; Figure 5 The effect of Lactobacillus plantarum LWQ17 CFS on the conductivity of Bacillus species, where A is Bacillus licheniformis and B is Bacillus amyloliquefaciens. Figure 6 The biofilm inhibition rate of Lactobacillus plantarum LWQ17 CFS against Bacillus subtilis; Figure 7 The effect of LWQ17 CFS on Bacillus plantarum on cell membrane permeability of Bacillus was investigated. A and C were the control groups for Bacillus licheniformis and Bacillus amyloliquefaciens, respectively, while B and D were the LWQ17 CFS treatment groups for Bacillus licheniformis and Bacillus amyloliquefaciens, respectively. Figure 8 The effects of different pretreatment methods on the sensory evaluation of steamed buns. Detailed Implementation

[0023] To better illustrate the objectives, technical solutions, and advantages of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0025] Example 1: Isolation and identification of spoilage bacteria in spoiled steamed buns under high summer temperatures 1. Isolation and identification of putrefactive bacteria Under aseptic conditions, 10 g of spoiled steamed buns grown at high summer temperatures (45℃) were weighed into a homogenizing bag containing 90 mL of 0.85% (v / v) sterile physiological saline and homogenized. The sample solution was then serially diluted with 0.85% (v / v) sterile physiological saline. 100 μL of each diluted solution was spread onto LB agar plates and incubated at 37℃ for 48 h. The colony morphology of the strains on the plates was observed to identify spoilage bacteria. The labeled colonies were isolated and purified using the streak plate method until no other bacteria were present on the LB agar plates. The purified strain plates were stored at 4℃ for later use.

[0026] This invention isolated 34 representative bacterial colonies with different morphologies from spoiled steamed buns in summer. After purification, 16S rRNA gene sequencing was performed, and the 34 spoilage bacteria were preliminarily identified as belonging to the genus Bacillus. Based on bacterial morphology ( Figure 1 Based on the bacterial characteristics (Table 1), they were divided into 10 groups, and one strain from each group was selected for physiological and biochemical analysis. Since Bacillus subtilis and Bacillus amyloliquefaciens are highly similar in physiological and biochemical characterization, the gyrB gene of strains Y-2, Y-6, Y-9, and Y-10 was further sequenced.

[0027] The results of 16S rRNA sequencing, physiological and biochemical identification, and gyrB gene sequencing (Table 2) show that four Bacillus species were screened from spoiled steamed buns under high temperature and humidity conditions in summer: Bacillus licheniformis (38.2%), Bacillus amyloliquefaciens (32.4%), Bacillus subtilis (23.5%), and Bacillus megaterium (5.9%). Bacillus licheniformis and Bacillus amyloliquefaciens, which had the highest abundance in the spoiled steamed buns, were selected. The phylogenetic trees of strains Y-8 and Y-9 are shown below. Figure 2 As shown, Bacillus licheniformis and Bacillus amyloliquefaciens were selected for subsequent experiments. The Bacillus licheniformis and Bacillus amyloliquefaciens used in the subsequent experiments were obtained through commercial channels.

[0028] Table 1. Colony characteristics of putrefactive bacteria .

[0029] Note: The percentage (%) is calculated as follows: (Number of strains in each group / Total number of strains) × 100% Table 2. Identification results of Bacillus.

[0030] Example 2: Sources and screening of lactic acid bacteria that inhibit the growth of Bacillus. The *Pediococcus pentosaceus* LWQ1 described in this invention is deposited at the China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; deposit date: September 27, 2023; the accession number for *Pediococcus pentosaceus* LWQ1 is CCTCC NO: M 20231802.

[0031] The Lactiplantibacillus plantarum LWQ17 described in this invention is deposited at the China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; deposit date: September 27, 2023; the accession number for Lactiplantibacillus plantarum LWQ17 is CCTCC NO: M 20231803.

[0032] The strains of Pediococcus pentosaceus LWQ7, LWQ9, LWQ12, LWQ13, LWQ14, LWQ16, and LWQ19 were all obtained from commercially available sources.

[0033] The strains *Lactobacillus plantarum* LWQ2, LWQ3, LWQ4, LWQ5, LWQ6, LWQ8, LWQ10, LWQ11, LWQ15, LWQ18, and LWQ20 were all obtained through commercial channels.

[0034] Preparation of fermentation supernatant (CFS): The lactic acid bacteria strain preserved in glycerol tubes was thawed, activated twice, centrifuged at 8000 r / min for 5 min at 4℃, the bacterial sludge was discarded, and the supernatant was obtained by filtration through a 0.22 μm filter membrane and stored at 4℃ for later use.

[0035] As shown in Example 1, Bacillus licheniformis is one of the main microorganisms causing spoilage of steamed buns in summer. Therefore, Bacillus licheniformis was used as an indicator bacterium, and the inhibitory effect of laboratory lactic acid bacteria strains on Bacillus licheniformis was determined by the 96-well plate method (Table 3). Under acidic conditions, lactic acid bacteria showed good inhibitory effects on Bacillus licheniformis, with inhibition rates greater than 84% after 12 h of co-cultivation and greater than 90% after 24 h of co-cultivation. Under neutral conditions, the inhibitory effect of lactic acid bacteria on Bacillus licheniformis weakened, but after 24 h of co-cultivation, the inhibition rates of Pediococcus pentosaceus LWQ13 and LWQ16 were greater than 78%, and the inhibition rates of Lactobacillus plantarum LWQ17, LWQ18, and LWQ20 were greater than 82%.

[0036] Table 3. Inhibition rates of different lactic acid bacteria and pH values ​​against Bacillus licheniformis , .

[0037] Note: "-" indicates no antibacterial effect. Different lowercase letters in the same column indicate significant differences between *Pediococcus pentosaceus* (p<0.05); different uppercase letters in the same column indicate significant differences between *Lactobacillus plantarum* (p<0.05).

[0038] The five lactic acid bacteria strains (LWQ13, LWQ16, LWQ17, LWQ18, and LWQ20) were quantitatively screened twice using the Oxford cup agar diffusion method, and lactic acid bacteria strains that showed good inhibitory effects on both Bacillus licheniformis and Bacillus amyloliquefaciens were further screened out (Table 4).

[0039] Table 4. Inhibitory effects of different lactic acid bacteria on Bacillus licheniformis and Bacillus amyloliquefaciens .

[0040] Note: Different lowercase letters in the same column indicate significant differences in lactic acid bacteria under acidic conditions (p<0.05); different uppercase letters in the same column indicate significant differences in lactic acid bacteria under slightly acidic conditions (p<0.05).

[0041] Under acidic conditions, lactic acid bacteria showed good inhibitory effects against Bacillus licheniformis and Bacillus amyloliquefaciens. Among them, LWQ17 exhibited the best antibacterial effect. After co-culturing LWQ17 CFS with Bacillus licheniformis for 10 h, the diameter of the inhibition zone was 22.9±0.1 mm, and after co-culturing with Bacillus amyloliquefaciens for 10 h, the diameter of the inhibition zone was 20.7±0.1 mm. Under slightly acidic conditions, Lactobacillus plantarum LWQ17 still showed better antibacterial effects than other strains.

[0042] Example 3: Study on the mechanism of Lactobacillus plantarum LWQ17 inhibiting the activity of Bacillus subtilis The LWQ17 CFS described in this embodiment was prepared in Example 2.

[0043] 1. Effects on morphological changes of Bacillus Scanning electron microscopy (SEM) allows for a clearer observation of the morphological changes of Bacillus licheniformis and Bacillus amyloliquefaciens after treatment with LWQ17 CFS.

[0044] Bacillus licheniformis and Bacillus amyloliquefaciens were cultured in a shaker until they reached the logarithmic growth phase. Then, 20 mL of LWQ17CFS was added, and the culture was continued in a shaker (37℃, 180 r / min) for 10 h. A control group without fermentation supernatant was used. The cultured bacterial suspension was centrifuged at 5000 r / min for 10 min, and the cells were washed twice with sterile PBS buffer, discarding the supernatant. An equal volume of 2.5% glutaraldehyde was added, and the suspension was fixed overnight at 4℃. After fixation, the cells were washed 2-3 times with sterile PBS buffer, dehydrated using an ethanol gradient (30%, 50%, 70%, 80%, 90%) for 15 min, and finally dehydrated with anhydrous ethanol for 15 min, repeated twice. The samples were critically dried with carbon dioxide and then sputter-coated with gold. The samples were observed under a scanning electron microscope at 9000 magnification at 5 kV and 10 μA.

[0045] Combination Figure 3 As shown, the control group ( Figure 3 The bacterial cells (as shown in A and C) have intact structures, smooth surfaces, and normal rod-shaped bacterial morphology. After treatment with LWQ17 CFS ( Figure 3 As shown in B and D, the bacterial cell surface is rough and wrinkled, with irregular shape, and the cells show phenomena such as aggregation, invading, or decomposition and breakage. Changes in bacterial cell morphology are also one of the reasons for the reduction or loss of bacterial vitality.

[0046] In existing technologies, Bacillus cereus cells treated with rosmarinic acid exhibit shrinkage and collapse, indicating that rosmarinic acid inhibits bacterial growth by disrupting the Bacillus cereus cell membrane. Furthermore, treatment with sucrose laurate and nisin damages the Bacillus subtilis cell membrane structure, leading to cell shrinkage and deformation. Therefore, combining the aforementioned morphological changes, it can be concluded that LWQ17 CFS causes osmotic pressure imbalance between the inside and outside of the cell by disrupting cell membrane integrity, resulting in cell rupture and ultimately bacterial death.

[0047] 2. The effect of Lactobacillus plantarum LWQ17 on the leakage of Bacillus nucleic acid Damage to the cell membrane increases membrane permeability, causing small molecules such as ions to leak out of the cell, followed by the release of large molecules such as nucleic acids. To investigate the effect of *Lactobacillus plantarum* LWQ17 on the cell membrane integrity of *Bacillus*, changes in cellular nucleic acid leakage were measured over 12 hours.

[0048] The release of intracellular macromolecules absorbed at 260 nm was assessed. *Bacillus licheniformis* and *Bacillus amyloliquefaciens* were cultured in a shaker to the logarithmic growth phase, then 20 mL of LWQ17 CFS was added, and the culture was continued in a shaker (37℃, 180 r / min) for 12 h. Samples were taken every 2 h to measure nucleic acid leakage, with sterile distilled water as a control. The absorbance at 260 nm was measured using an ultra-micro spectrophotometer, with three replicates per group.

[0049] from Figure 4 As can be seen, after treatment with LWQ17 CFS, the absorbance values ​​of the two Bacillus strains measured at 260 nm increased rapidly within 0–10 h, and then the growth slowed down and tended to stabilize. This indicates that LWQ17 CFS disrupts the integrity of the Bacillus cell membrane, leading to more contents permeating into the external fluid.

[0050] 3. Effect of Lactobacillus plantarum LWQ17 on the conductivity of Bacillus subtilis Electrical conductivity values ​​reflect changes in cell membrane permeability. As a crucial protective barrier for cells, the cell membrane's integrity is essential for maintaining intracellular homeostasis. When exposed to antimicrobial substances, the cell membrane structure is damaged, leading to loss of barrier function and subsequent leakage of intracellular electrolytes into the culture medium, thus increasing the medium's conductivity.

[0051] Bacillus licheniformis and Bacillus amyloliquefaciens cultured to the logarithmic growth phase were centrifuged at 5000 r / min for 10 min, the supernatant was discarded, and the precipitate was washed three times with sterile PBS buffer and resuspended. LWQ17 CFS was added, with sterile distilled water as the control group. The samples were incubated on a shaker (37°C, 180 r / min) for 12 h. Samples were taken every 2 h, centrifuged at 5000 r / min for 10 min, and the conductivity of the supernatant was measured using a conductivity meter. Each group was repeated three times and the average value was taken.

[0052] Combined with appendix Figure 5 As shown, with the increase of culture time, the conductivity values ​​of the two strain suspensions were significantly higher than those of the control group, and showed an increasing trend with the increase of treatment time. When the culture time was 10-12 h, there was no significant difference in the change of conductivity values. This indicates that LWQ17 CFS can increase the cell membrane permeability of Bacillus licheniformis and Bacillus amyloliquefaciens, causing leakage of cell contents.

[0053] 4. Biofilm inhibition rate of Lactobacillus plantarum LWQ17 against Bacillus subtilis Bacillus licheniformis and Bacillus amyloliquefaciens cultured to the logarithmic growth phase were mixed, and 100 μL of each culture and 100 μL of Lactobacillus plantarum LWQ17 CFS were added to 96-well plates, with a control group without fermentation supernatant. The 96-well plates were incubated on a shaker (37°C, 180 r / min) for 10 h. After pouring out the bacterial suspension, each well was washed three times with sterile PBS buffer to remove loosely attached cells and dried at room temperature. 200 μL of methanol was added to each well to fix firmly attached biofilm cells, the liquid was discarded, and the plates were dried at room temperature. 200 μL of 0.1% (w / v) crystal violet was added to each well for staining for 10 min, the crystal violet staining was poured out, and each well was washed three times with sterile PBS to remove excess staining and dried at room temperature. 200 μL of 33% (v / v) glacial acetic acid was added to each well for treatment for 10 min. The sample solutions were measured at 570 nm (OD500) using a multi-mode microplate reader. 570 The absorbance value was measured at ( ).

[0054] Biomembrane inhibition rate (%) = [(OD 对照 - OD 处理 ) / OD 对照 ] × 100 The biofilm inhibition rate of *Lactobacillus plantarum* LWQ17 against two Bacillus strains is as follows: Figure 6As shown, *Lactobacillus plantarum* LWQ17 inhibited *Bacillus licheniformis* by 81.2 ± 4.13% and *Bacillus amyloliquefaciens* by 69.2 ± 8.30%. This indicates that *Lactobacillus plantarum* LWQ17 can alter the surface properties of the two Bacillus strains and prevent them from aggregating, thereby inhibiting biofilm formation.

[0055] 5. Effect of Lactobacillus plantarum LWQ17 on Bacillus cell membrane permeability (PI staining) Propidium iodide (PI) cannot penetrate cells with intact plasma membranes. However, when the cell membrane is damaged, it can enter the cell and specifically bind to nucleic acids, thereby generating a red fluorescent signal.

[0056] Centrifuge the cultured bacterial solution at 5000 r / min for 10 min, wash the bacterial cells twice with sterile PBS buffer and discard the supernatant, add PI staining solution (20 μg / mL), mix well and stain at room temperature in the dark for 20 min, wash off excess dye 2 to 3 times with PBS, resuspend in PBS buffer and observe under a laser confocal microscope.

[0057] Combination Figure 7 Observations revealed that no red fluorescence was observed in the control group, while significant red fluorescent areas were observed in both Bacillus licheniformis and Bacillus amyloliquefaciens treated with LWQ17 CFS. This phenomenon indicates that LWQ17 CFS treatment of Lactobacillus plantarum damages the Bacillus cell membrane and increases cell membrane permeability.

[0058] Example 4: The effect of different pretreatment methods on the shelf life of steamed buns Based on different steamed bun recipes, they are divided into 5 groups: CK group, LWQ17 group, LWQ1 group, LWQ20 group, and *Lactobacillus reuteri* group. (The shelf life of the steamed buns described in this invention is defined as the day before the first observation of spoilage bacteria, and the *Lactobacillus reuteri* is obtained through commercial channels.)

[0059] Preparation of sourdough for different groups: The bacterial strains corresponding to different groups were activated twice in MRS broth, anaerobically cultured at 37℃ for 24 h, centrifuged at 8000 r / min for 3 min to harvest the culture, and washed twice with 0.1% peptone water for later use. 2 mL of bacterial culture (10... 9 Mix (cfu / mL) with 100 g wheat flour and 100 mL water, place in a fermentation chamber, and ferment at 34°C and 85%RH for 14 h to prepare the corresponding sourdough.

[0060] Steamed buns were prepared using a two-stage fermentation method. The ingredients for each group of steamed buns in Table 5 were mixed and kneaded until the dough surface was smooth. The dough was then placed in a fermentation box under the following conditions: 37℃, 85%RH, and fermented for 2 hours. The dough was then divided into 50g portions, rounded, and shaped. The buns were allowed to rise at room temperature for 30 minutes, steamed for 30 minutes, and then cooled to room temperature under ultraviolet light.

[0061] The steamed buns were sealed in food-grade polyethylene bags. The sealed steamed buns were then placed under different pretreatment conditions (30°C~50°C, 40%~85%RH constant temperature and humidity chambers for 12~24 h). The pretreated steamed buns were then placed under different storage conditions (simulating room temperature or summer high temperature). The surface changes of each group of steamed buns were observed, and the spoilage time of the steamed buns was recorded (recorded as the germination time of microorganisms (mold or Bacillus)).

[0062] Table 5 Steamed Bun Recipe Note: "-" indicates that it has not been added.

[0063] Based on different treatment methods, the steamed buns were divided into four groups: a 12-hour group, a 24-hour group, a continuous treatment group, and a room temperature group. "12-hour" means the steamed buns were pre-treated at high temperature (30-50℃) for 12 hours after steaming and then placed at room temperature for observation; "24-hour" means the steamed buns were pre-treated at high temperature (30-50℃) for 24 hours after steaming and then placed at room temperature for observation; "continuous" means the steamed buns were kept in a high-temperature (30-50℃) constant temperature and humidity chamber after steaming; and "room temperature" means the steamed buns were kept at room temperature (22-37°C, subject to the actual temperature markings for each group). Five samples were placed in each group, and the surface changes of the samples were observed for 10 days under different conditions, and the germination time of spoilage bacteria was recorded.

[0064] 1. Effect of 37℃ and 65%RH pretreatment on the shelf life of steamed buns at room temperature (room temperature storage conditions: 35 ~ 37℃, 80 ~ 90%RH) As shown in Table 6, spoilage bacteria were first observed in the CK steamed buns at room temperature on day 2. Pretreatment at 37℃ and 65% RH extended the shelf life of CK steamed buns by one day. Spoilage bacteria were first observed in the LWQ17 steamed buns at room temperature on day 3. Pretreatment at 37℃ and 65% RH extended the shelf life by 2-3 days. Spoilage bacteria were first observed in the LWQ1 steamed buns at room temperature on day 3. Pretreatment at 37℃ and 65% RH extended the shelf life by one day. Spoilage bacteria were first observed in the LWQ20 steamed buns at room temperature on day 3. Pretreatment at 37℃ and 65% RH extended the shelf life by 1-2 days. Spoilage bacteria were first observed in all steamed buns containing *Lactobacillus reuteri* at room temperature on day 3. Pretreatment at 37℃ and 65% RH extended the shelf life by 1-2 days.

[0065] The steamed buns prepared from lactic acid bacteria sourdough (lactoic acid bacteria LWQ17, LWQ1, LWQ20 and Lactobacillus reuteri) were pretreated (37℃, 65%RH, for more than 12 hours), and their shelf life was extended by 2 to 3 days compared with the CK steamed buns at actual room temperature (35 ~ 37℃, 80 ~ 90%RH).

[0066] Table 6. Microbial germination time of steamed buns after pretreatment at 37℃ and 65%RH for 10 days of storage. Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0067] 2. Effect of 45℃ and 85%RH pretreatment on the shelf life of steamed buns at room temperature (room temperature storage conditions: 30 ~ 35℃, 70 ~ 80%RH) As shown in Table 7, spoilage bacteria were first observed in the CK steamed bun room temperature group on day 2, and the shelf life was extended by 1 day after pretreatment at 45℃ and 85%RH. Spoilage bacteria were first observed in the LWQ17 steamed bun room temperature group on day 4, and the shelf life was extended by 6 days after pretreatment at 45℃ and 85%RH. Spoilage bacteria were first observed in the LWQ1 steamed bun room temperature group on day 3, and the shelf life was extended by 2-4 days after pretreatment at 45℃ and 85%RH. Spoilage bacteria were first observed in the LWQ20 steamed bun room temperature group on day 3, and the shelf life was extended by 2-4 days after pretreatment at 45℃ and 85%RH. Spoilage bacteria were first observed in the Lactobacillus reuteri steamed bun room temperature group on day 3, and the shelf life was extended by 2 days after pretreatment at 45℃ and 85%RH.

[0068] Steamed buns prepared from lactic acid bacteria sourdough (with lactic acid bacteria LWQ17, LWQ1, LWQ20 and Lactobacillus reuteri) were pretreated (at 45°C and 85%RH for more than 12 hours). The shelf life of the steamed buns was extended by 3 to 8 days compared to the CK steamed buns at actual room temperature (30-35°C and 70-80%RH).

[0069] Table 7. Microbial germination time of steamed buns after pretreatment at 45℃ and 85%RH for 10 days of storage. Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0070] 3. Effect of 30℃ and 65%RH pretreatment on the shelf life of steamed buns at room temperature (room temperature storage conditions: 22 ~ 25℃, 40 ~ 60%RH) As shown in Table 8, spoilage bacteria were first observed in both the CK steamed buns (room temperature and 12 h groups) on the second day. Pretreatment at 30℃ and 65%RH for more than 24 h extended the shelf life by one day. Spoilage bacteria were first observed in the LWQ17 steamed buns (room temperature group) on the third day. Pretreatment at 30℃ and 65%RH extended the shelf life by two days. Spoilage bacteria were first observed in the LWQ1 steamed buns (room temperature group) on the second day. Pretreatment at 30℃ and 65%RH extended the shelf life by 1-2 days. Spoilage bacteria were first observed in both the LWQ20 steamed buns (room temperature and 12 h groups) on the third day. Pretreatment at 30℃ and 65%RH for more than 24 h extended the shelf life by one day. Spoilage bacteria were first observed in both the Lactobacillus reuteri steamed buns (room temperature and 12 h groups) on the third day. Pretreatment at 30℃ and 65%RH for more than 24 h extended the shelf life by one day.

[0071] Steamed buns prepared from lactic acid bacteria sourdough (with lactic acid bacteria LWQ17, LWQ1, LWQ20 and Lactobacillus reuteri) were pretreated (30℃, 65%RH, for more than 12 hours). The shelf life of the steamed buns was extended by 1 to 3 days compared with the CK steamed buns at actual room temperature (22~25℃, 40~60%RH).

[0072] Table 8. Microbial germination time of steamed buns after pretreatment at 30℃ and 65%RH for 10 days of storage. Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0073] 4. Effect of 37℃ and 85%RH pretreatment on the shelf life of steamed buns at room temperature (room temperature storage conditions: 22 ~ 25℃, 40 ~ 60%RH) As shown in Table 9, spoilage bacteria were first observed in the CK room temperature group steamed buns on the second day. After pretreatment at 37℃ and 85% RH, the shelf life was extended by 1 day. Spoilage bacteria were first observed in the LWQ17 room temperature group steamed buns on the third day. After pretreatment at 37℃ and 85% RH, the shelf life was extended by 7 days. Spoilage bacteria were first observed in the LWQ1 room temperature group steamed buns on the third day. After pretreatment at 37℃ and 85% RH, the shelf life was extended by 1-3 days. Spoilage bacteria were first observed in the LWQ20 room temperature group steamed buns on the third day. After pretreatment at 37℃ and 85% RH, the shelf life was extended by 2-4 days. Spoilage bacteria were first observed in the Lactobacillus reuteri steamed buns on the third day. After pretreatment at 37℃ and 85% RH, the shelf life was extended by 1 day.

[0074] Steamed buns prepared from lactic acid bacteria sourdough (with lactic acid bacteria LWQ17, LWQ1, LWQ20 and Lactobacillus reuteri) were pretreated (at 37°C, 85%RH, for more than 12 hours). The shelf life of the steamed buns was extended by 2 to 8 days compared to the CK steamed buns at actual room temperature (22~25°C, 40~60%RH).

[0075] Table 9. Microbial germination time of steamed buns after pretreatment at 37℃ and 85%RH for 10 days of storage. Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0076] 5. Effect of 50℃ and 85%RH pretreatment on the shelf life of steamed buns at room temperature (room temperature storage conditions: 22 ~ 25℃, 40 ~ 60%RH) As shown in Table 10, spoilage bacteria were first observed in the CK (room temperature group) and continuous treatment group steamed buns on the second day. After 12h and 24h treatments, the shelf life was extended by 1 day. Spoilage bacteria were first observed in the LWQ17 steamed bun room temperature group on the third day. After pretreatment at 50℃ and 85%RH, the shelf life was extended by 7 days. Spoilage bacteria were first observed in the LWQ1 steamed bun room temperature group on the third day. After pretreatment at 50℃ and 85%RH, the shelf life was extended by 2-3 days. Spoilage bacteria were first observed in the LWQ20 steamed bun room temperature group on the third day. After pretreatment at 50℃ and 85%RH, the shelf life was extended by 3-4 days. Spoilage bacteria were first observed in the Lactobacillus reuteri steamed bun room temperature group on the third day. After pretreatment at 50℃ and 85%RH, the shelf life was extended by 1-2 days.

[0077] Steamed buns prepared from lactic acid bacteria sourdough (with lactic acid bacteria LWQ17, LWQ1, LWQ20 and Lactobacillus reuteri) were pretreated (at 50°C and 85%RH for more than 12 hours). The shelf life of the steamed buns was extended by 2 to 8 days compared to the CK steamed buns at actual room temperature (22-25°C and 40-60%RH).

[0078] Table 10 Microbial germination time of steamed buns after pretreatment at 50℃ and 85%RH for 10 days of storage Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0079] 6. The effect of high temperature and high humidity pretreatment on the shelf life of steamed buns stored at high temperature and high humidity Based on the experimental results of pretreatment and storage at room temperature, the optimal LWQ17 sourdough was selected to prepare steamed buns. The shelf life of the steamed buns was then observed in conjunction with high-temperature and high-humidity pretreatment. Five samples were prepared in each group. First, the steamed buns prepared with LWQ17 sourdough were pretreated at 37-50℃ and 85%RH for 24 hours. Then, they were stored under high-temperature and high-humidity conditions (45℃, 85%RH). Changes on the surface of the steamed buns were observed, and the germination time of spoilage bacteria was recorded.

[0080] As shown in Table 11, Bacillus germination was first observed in the steamed buns of the CK group on the 3rd day (the shelf life was extended by 1 day compared to the CK room temperature group). After pretreatment at 37℃, Bacillus germination was first observed in the steamed buns of the LWQ17 group on the 6th day, extending the shelf life by 3 days compared to the CK group; after pretreatment at 45℃ and 50℃, no spoilage bacteria germination was observed within 10 days, extending the shelf life by 7 days compared to the CK group.

[0081] Table 11 Microbial germination time of steamed buns after high temperature and high humidity pretreatment and storage for 10 days Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0082] 7. The effect of high temperature and low humidity pretreatment on the shelf life of steamed buns stored at high temperature and high humidity (45℃, 85%RH) As shown in Table 12, Bacillus germination was first observed in the steamed buns of the CK group on the 3rd day. After pretreatment at 37℃, Bacillus germination was first observed in the steamed buns of the LWQ17 group on the 5th day, extending the shelf life by 2 days compared to the CK group; after pretreatment at 45℃ and 50℃, no spoilage bacteria germination was observed within 10 days, extending the shelf life by 7 days compared to the CK group.

[0083] Table 12 Microbial germination time of steamed buns after high temperature and low humidity pretreatment and storage for 10 days Note: The numbers in the table represent the different types of putrefactive bacteria observed for the first time: Arabic numerals represent Bacillus, lowercase Chinese numerals represent mold, and uppercase Chinese numerals represent both Bacillus and mold.

[0084] Example 5: The effect of pretreatment on the sensory evaluation of steamed buns The sensory evaluation group consisted of 10 food science students (five men and five women) who were sensitive to taste intensity, had no food allergies or intolerances, and regularly conducted formal sensory evaluations. The participants evaluated the steamed buns' surface color, appearance, texture, aroma, mouthfeel, elasticity, stickiness, and chewiness. Specific scoring criteria are detailed in Table 13.

[0085] Table 13 Sensory Evaluation Criteria for Steamed Buns After pretreatment, the steamed buns in each group were subjected to sensory evaluation, and the results are as follows: Figure 8 The steamed buns from the CK 12 h group had the best surface color, followed by the LWQ17 12 h group, with other groups scoring similarly. After 12 h and 24 h treatments, the appearance and texture of the steamed buns were superior to those from the room temperature group. The steamed buns from the LWQ17 12 h, LWQ17 24 h, and CK 12 h treatment groups had better aromas than other groups, with the LWQ17 room temperature group scoring the lowest. The steamed buns from the LWQ17 12 h treatment group had the best texture and elasticity, while the LWQ17 room temperature group had the worst texture and elasticity. The steamed buns from the LWQ17 24 h treatment group had the highest stickiness. The CK 12 h group had the best chewiness, while the CK room temperature group and the LWQ17 room temperature group had relatively poor chewiness.

[0086] In summary, considering the pretreatment of steamed buns at different temperatures and humidity levels, all pretreatments extended the shelf life of the steamed buns compared to those at room temperature. The LWQ17 group showed the most significant shelf-life extension, extending it by 6-7 days after pretreatment at higher temperatures and humidity (45℃, 85%RH and 37℃, 85%RH). The LWQ20 and LWQ1 groups showed the second most significant extensions, both extending the shelf life by 2-4 days after pretreatment at 37℃ and 85%RH. The *Lactobacillus reuteri* group showed a relatively stable shelf-life extension, extending it by 1-2 days under different pretreatment conditions. While its effect was weaker than the LWQ17, LWQ1, and LWQ20 groups, it was consistently better than the CK group. The CK group had the shortest shelf life, extending it by only 1 day under all pretreatment conditions. After undergoing high temperature and low humidity, and high temperature and high humidity pretreatment followed by high temperature and high humidity storage, the LWQ17 group of steamed buns still showed the most significant effect in extending the shelf life, which could be extended by 7 days.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. Application of Lactobacillus plantarum LWQ17 in the preparation of biological agents to inhibit spoilage bacteria in steamed buns.

2. The application according to claim 1, characterized in that, The preservation number of the Lactobacillus plantarum LWQ17 is CCTCC NO: M 20231803.

3. The application according to claim 1, characterized in that, The bacteria causing the spoilage of the steamed buns include Bacillus licheniformis and / or Bacillus amyloliquefaciens.

4. The application according to claim 1, characterized in that, The plant lactobacillus LWQ17 can disrupt the cell membrane integrity of Bacillus and increase the cell membrane permeability of Bacillus; the plant lactobacillus LWQ17 can destroy the bacterial cells of Bacillus, making their surface rough and wrinkled, irregular in shape, and causing cells to aggregate, invaginate, or decompose and break down.

5. The application according to claim 1, characterized in that, The specific ways in which *Lactobacillus plantarum* LWQ17 inhibits spoilage bacteria in steamed buns include: sealing the steamed buns prepared by fermentation with *Lactobacillus plantarum* LWQ17, and then pre-treating the sealed steamed buns to extend their shelf life.

6. The application according to claim 5, characterized in that, The pretreatment conditions are a temperature of 30 to 50°C, a humidity of 40% to 85%, and a pretreatment time of 12 to 24 hours.

7. The application according to claim 1, characterized in that, The steamed buns should be stored at 22-50℃.

8. The application according to claim 1, characterized in that, The steamed buns can be stored for 5 to 10 days.

9. The application according to claim 1, characterized in that, Based on the mass of flour, the bacterial content of *Lactobacillus plantarum* LWQ17 in the preparation of steamed buns is 10. 7 ~ 10 9 cfu / g.

10. Application of Lactobacillus plantarum LWQ17 in inhibiting Bacillus.

Images