Bacteriocin ZJQ2-1 produced by Pediococcus pentosaceus from traditional sweet wine koji: purification, characterization, and application in fermented glutinous rice

doi:10.21203/rs.3.rs-6341859/v1

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Bacteriocin ZJQ2-1 produced by Pediococcus pentosaceus from traditional sweet wine koji: purification, characterization, and application in fermented glutinous rice

https://doi.org/10.21203/rs.3.rs-6341859/v1

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published 22 Jul, 2025

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Background: Fermented glutinous rice has low alcohol content and abundant nutrients, and it is easy to be contaminated by Bacillus cereus during production and preservation. This study aimed to inhibit the spoilage bacterium Bacillus cereus in the fermented glutinous rice by bacteriocin-producing lactic acid bacteria and its bacteriocin.

Results: Bacteriocin-producing Pediococcus pentosaceus JQ2-1 was isolated from traditional sweet wine koji and its bacteriocin ZJQ2-1 was purified by ethyl acetate extraction, cation exchange chromatography, gel chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC). Bacteriocin ZJQ2-1 was identified by HPLC-MS/MS, and the results showed it had a molecular weight of 1204.43 Da and the amino acid sequence was KIGLFGGAGVGKT. Further characterization analysis showed that the bacteriocin ZJQ2-1 was thermostable (30 min, 121°C), highly pH stable (2-10), sensitive to protease and exhibited broad-spectrum antibacterial ability against Gram-positive and Gram-negative bacteria. The count of Bacillus cereusin the fermented glutinous rice that was inoculated with P. pentosaceusJQ2-1 and bacteriocin ZJQ2-1 decreased by about 80% and 64% of that in the initial stage, respectively.

Conclusions: These results indicated the potential of P. pentosaceus JQ2-1 and bacteriocin ZJQ2-1 as a bio-preservative in glutinous rice products.

sweet wine koji

bacteriocin

purification

fermented glutinous rice

Fermented glutinous rice, also known as rice wine, is made of rice fermented by sweet wine koji, has a long history in many Asian countries (Lv, Huang, Zhang, Rao, & Ni, 2013). As a kind of conventional alcoholic beverage, fermented glutinous rice has achieved a high level of popularity in China, owing to its distinctive flavour, low alcohol content, and abundance of nutrients such as amino acids, peptides, vitamins, and oligosaccharides.(Qing Ren, Sun, Wu, Wang, Wang, Zheng, et al., 2019). However, fermented glutinous rice has a low alcohol content and is rich in nutrients, during production and storage, it is susceptible to contamination from Escherichia coli, Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella, and Staphylococcus aureus, which can affect its quality and shelf life. Among which, Bacillus cereus is the most common spoilage bacterium (Kim, Jun, Lee, Hwang, & Rhee, 2018; Ruiling, Thunthacha, Mingming, Man, Jianwei, Tian, et al., 2018; Suzuki, Asano, Iijima, & Kitamoto, 2008).

In order to extend the shelf life of the fermented glutinous rice, it is customary to use a combination of locally-grown medicinal plants, herbs and spices as food additives. The purpose of this is to inhibit the growth of any potentially harmful microorganisms during the fermentation process. For example, Murugan et al. found that lotus and goat grass can enhance antibacterial activity against Staphylococcus aureus in rice wine (Murugan, Mishra, & Paul, 2018). Choi et al. found that grape seed extract with a concentration of 0.1–0.2% can reduce the concentration of bacteria and yeast of bottled fresh rice wine (Choi, Yeo, Choi, & Jeong, 2016). However, plant additives often reduce the vitality of starter culture, leading to a lower rice wine quality. Heat treatment is another a well-known method for rice wine preservation, which is economically feasible and effective in inactivating pathogenic microorganisms. However, thermal processing of rice wine inevitably led to the loss of volatile alcohols and aldehydes, resulting in low overall acidity (Yang, Xia, Wang, Tao, Yu, & Ai, 2019). Therefore, new sterilization methods should be developed to extend the shelf life of rice wine.

Bacteriocins produced by lactic acid bacteria are peptide substances, which are non-toxic to eukaryotic cells such as yeast, easily digested by the human gastrointestinal tract, thermally stable, effective against food spoilage and pathogenic bacteria at low concentrations, therefore, they can be used as food preservatives(Galvez, Abriouel, Lopez, & Ben Omar, 2007; Johnson, Jung, Jin, Jayabalan, Yang, & Suh, 2018). In this study, the bacteriocin-producing lactic acid bacterium was screened from traditional sweet wine koji. The bacteriocin was purified and identified by HPLC-MS/MS combined with genome sequencing of the producing lactic acid bacterium. The stability and antibacterial spectrum of bacteriocin were studied. In addition, the inhibitory effects of lactic acid bacterium and bacteriocin against spoilage organisms was investigated during the rice wine fermentation and preservation. This study provides technical support for using bacteriocins producing lactic acid bacteria to extend the shelf life of rice wine.

2.1. Screening and identification of bacteriocin-producing lactic acid bacteria

2.1.1. Isolation of bacteriocin-producing lactic acid bacteria

Samples of the traditional sweet wine koji were gathered from various Chinese areas. After crushing 5g of traditional sweet wine koji, it was submerged in 45mL of sterile 0.9% NaCl solution and homogenized for 10 minutes in a homogenizer (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China). A MRS agar plate containing 3% calcium carbonate was inoculated with 100 µL dilutions of the homogenate that had been continuously diluted tenfold with 0.9% NaCl solution. After that, the plates were incubated for 48 h at 37°C. (Hu, Zhao, Zhang, Yu, & Lu, 2013). The single colony with calcium dissolving ring were selected and incubated in MRS medium at 37°C for 48 h.

Lactic acid bacteria were cultured in MRS broth at 37°C for 24 h, and the cell-free fermentation supernatant (CFS) was collected by centrifugation (4°C, 8000 rpm/min, 15 min) (Centrifuge, Thermo-science, Germany). The CFS was then filtered through a 0.22 µm filter. The CFS was adjusted to pH 6 using 5 M NaOH with the purpose of eliminating the possibility of organic acid-induced antimicrobial activity. The antibacterial activity of CFS was determined by agar diffusion method using Bacillus cereus JN1L as indicator bacteria (Lei, Zhao, Sun, Ran, Ruan, & Zhu, 2020). To remove the interference of hydrogen peroxide, catalase solution (1 mg/mL) (Solarbio Science & Technology Co., Ltd., Beijing, China) was added to the CFS adjusted to pH 6 and left at 37°C for 30 min. After treatment, the antibacterial activity was measured and same CFS without catalase was used as control group. In order to confirm the antibacterial activity of the bacteriocin, the treated CFS were supplemented with trypsin and pepsin (1 mg/mL), respectively, at 37°C for 30 min. As a control, identical CFS were used without the addition of the corresponding enzyme (Qingxia Ren, Zhang, Xue, Liu, Yang, & Yang, 2023).

2.1.2. Identification of strain

A Gram staining kit (Changde BKMAM Trading Co., Ltd., China) was used to gram-stain the strain JQ2-1, and the morphology was examined under a light microscope. Strain JQ2-1 DNA was extracted according to the method of Bacterial Genomic DNA Extraction Kit (Sangon Biotech Co., Ltd, China), and then amplified by PCR using universal primers 27F and 1492R. Following a five-minute pre-denaturation period at 94°C, the subsequent PCR amplification process entailed a series of thermal cycles comprising denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 50 seconds. A total of 35 cycles of these three phases were completed, followed by an extension at 72°C for 10 min (Pei, Jin, Abd El-Aty, Baranenko, Gou, Zhang, et al., 2020). Following an examination by agarose gel electrophoresis, the PCR products were dispatched to Sangong Biotech Co., Ltd. (Shanghai, China) for Sanger sequencing. Phylogenetic trees were constructed using Molecular Evolutionary Genetics Analysis (MEGA), version 11.0.10, after the sequences were sent to NCBI for homology analysis (Tamura, Stecher, & Kumar, 2021).

2.2. Purification and identification of bacteriocin

2.2.1. Whole genome sequencing and annotation

The strain JQ2-1 was cultured in MRS broth at 37℃ for 12 h, and the cells were collected after centrifugation at 8000 rpm/min at 4℃ for 15 min. The collected cells were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for gene assembly and sequencing (Li, Liu, Zhang, Xiong, Hong, Jia, et al., 2023). Gene information of P. pentosaceus JQ2-1 was analyzed using Illumina platform. Annotation of the obtained genes using gene annotation databases (Pfam, KEGG, Swiss-Prot, GO, COG and NR) to obtain functional information about the genes. To find the genes encoding antimicrobial chemicals, genome mining was performed using the BAGEL4 and Uniport databases.

2.2.2. Purification of bacteriocin

CFS was extracted three times by mixing with equal volume of ethyl acetate. The upper layer of ethyl acetate was collected and the ethyl acetate was removed using a rotary evaporator (Shanghai Yarong Biochemical Instrument Co., Ltd., Shanghai, China). The agar diffusion method was used to determine the antibacterial activity of the ethyl acetate extract, with Bacillus cereus JN1L acting as indicator bacteria (Zhang, Yang, Liu, Wu, Fang, Wang, et al., 2020).

The ethyl acetate extract was subjected to cation exchange chromatography using a carboxymethyl cellulose 52 (CMC52) chromatography column (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China). Following equilibration with buffer A (20 mM sodium acetate, pH 4.5), crude bacteriocins were eluted with buffer B (20 mM sodium acetate, pH 4.5) containing a stepwise NaCl gradient (0 M, 0.1 M, 0.3 M, 1 M). The chromatographic separation was performed at 1.0 mL/min flow rate. Protein elution profiles were monitored by UV absorbance at 280 nm, with corresponding peak fractions collected for subsequent processing. The inhibitory activity of each elution peak fraction was determined by agar diffusion method (Ye, Wang, Liu, Li, & Gu, 2021).

The antibacterial active ingredient obtained from cation exchange chromatography was purified on a Sephadex G-25 column (Solarbio Science & Technology Co., Ltd., Beijing, China) equilibrated with ultrapure water. The column was eluted with ultrapure water at a flow rate of 1 mL/min until no absorption was detected at 280 nm. Each elution peak were collected and tested for antimicrobial activity using the agar diffusion method against the Bacillus cereus JN1L (Ye, Wang, Liu, Li, & Gu, 2021).

The active fractions underwent additional purification via reversed-phase high-performance liquid chromatography (RP-HPLC) employing a C18 analytical column (Waters, USA; 250 mm×19 mm i.d., 5 µm particle size). The mobile phase for chromatographic separation is composed of (A) 0.1% trifluoroacetic acid (TFA; Macklin, Shanghai) in ultrapure water and (B) 100% acetonitrile (ACN; Macklin, Shanghai). Sample (100 µL injection volume) was eluted at 2.0 mL/min with the following gradient profile: 10% B (0–10 min), linear increase to 20% B (10–20 min), followed by re-equilibration at 10% B (20–30 min) (Han, Zhang, Peng, Wu, & Zhong, 2023; Li, Guo, Zhang, Mu, Sha, Lin, et al., 2022). Elution peaks were collected at 280nm and acetonitrile was removed by rotary evaporation. The inhibitory activity of each elution peak was determined using agar diffusion method.

2.2.3. HPLC-MS/MS analysis of bacteriocin ZJQ2-1

The concentrated bacteriocin sample obtained after RP-HPLC was dispatched to Shanghai Fudan University for mass spectrometry analysis. The sample was subjected to hydrolysis by trypsin, after which the resulting peptide fragments was analysed using an Easy-nLC 1200 (Thermo Scientific, P/N LC140) and an Orbitrap Exploris 480 (Thermo Scientific, P/N BRE725533). Peptides were captured on a PepMap C18 column (100 µm×2 cm) at a flow rate of 10 µL/min for 3 min, after which they were separated by gradient elution on a PepMap C18 column (75 µm×25 cm). The MS employed Orbitrap for a preliminary scan, covering a range of 350–1600 (m/z) at a resolution of 120,000. The maximum ion introduction time was set at 50 milliseconds, and the top 20 parent ions that satisfied the higher energy C-trap dissociation (HCD) were utilised to break the MS/MS fragmentation conditions. The samples were then scanned with the Orbitrap at a resolution of 15,000. The minimum scan range was set at m/z = 110, and the scan range was automatically regulated based on the mass-charge ratio of the parent ion. It has been established that ions could be introduced for a maximum of 50 ms in MS/MS (Li, Yang, Li, Zheng, Xiong, Hou, et al., 2024). The amino acid sequences obtained by mass spectrometry were compared and identified using the NCBI and Uniport databases.

2.2.4. Molecular mass analysis of bacteriocin

The bacteriocin produced by P. pentosaceus JQ2-1 was named bacteriocin ZJQ2-1. The molecular weight of bacteriocin ZJQ2-1 was determined by Tricine SDS-PAGE analysis using discontinuous gels, including a 16.5% separating gel and a 4% stacking gel. Bacteriocin ZJQ2-1 and a low molecular weight marker were run at 80 V for 30 min and then at 120 V for the remainder of the separation. After electrophoretic separation, proteins were visualised by staining with Coomassie Brilliant Blue G-250 and destaining with ethyl alcohol acetic acid solution. The gel was photographed using the Gel Imaging System (Ye, Wang, Liu, Li, & Gu, 2021).

2.3. Characteristics of bacteriocin

2.3.1. Production kinetics of bacteriocin

The P. pentosaceus JQ2-1 was cultured in MRS broth medium at 37°C for 36 hours. Fermentation broth was taken every 3 h for measurement of pH and OD600 nm (Ruiz Martinez, Wachsman, Ignacio Torres, Guy LeBlanc, Todorov, & Gombossy de Melo Franco, 2013). Subsequent processing involved centrifugation (4℃, 8000 rpm/min, 15 min) to remove bacterial cells, followed by pH adjustment to 6.0 using sterile NaOH (5 M) to neutralize organic acid interference. The resulting cell-free supernatant was subsequently evaluated for antimicrobial efficacy against B. cereus JN1L through agar diffusion method (Qingxia Ren, Zhang, Xue, Liu, Yang, & Yang, 2023).

2.3.2. Antibacterial spectrum of bacteriocin ZJQ2-1

In order to investigate the antimicrobial spectrum of bacteriocin ZJQ2-1, several common food microorganisms were studied, including Gram-positive bacteria (Listeria monocytogenes ATCC 19116, Staphylococcus aureus CMCC 41002, Bacillus subtilis FS, SHOU, Staphylococcus saprophyticus FS, SHOU, Bacillus cereus Z-3), Gram-negative bacteria (Salmonella CMCC15611, Escherichia coli ATCC 25922, Vibrio parahaemolyticus ATCC 33847) and fungi (Wickerhamomyces anomalus J5-1 FS, SHOU, Cyberlindnera fabianii J21-1 FS, SHOU, Saccharomyces cerevisiae FS, SHOU, Rhizopus arrhizus M5-1 FS, SHOU) were used as indicator strains. All indicator bacteria tested in this study were obtained from the China Centre for Microbial Conservation and the Food Science Laboratory of Shanghai Ocean University. The measurement of antimicrobial activity was conducted by means of the agar diffusion method. To ensure the reliability of the results, each test was repeated thrice (Daba, Mostafa, Saleh, Elkhateeb, Awad, Nomiyama, et al., 2022).

2.3.3. Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of bacteriocin ZJQ2-1 against B. cereus JN1L was determined using the double dilution method (Cao, Du, Zhao, Xiao, Han, & Zhou, 2019). The indicator strain B. cereus JN1L was incubated in liquid LB medium for 12 hours, and then the bacteriocin ZJQ2-1 was serially diluted with sterile PBS (pH 7.2). A mixture of 10µL of bacteriocin dilution and 90µL of culture medium containing B. cereus JN1L (10⁶ CFU/mL) was added to a 96-well plate and co-cultured at 37℃ for 12 hours. Sterile PBS (pH 7.2) buffer was added as a control. A microplate reader (Thermo, Shanghai, China) was used to measure the absorbance of the B. cereus JN1L culture medium at 600 nm. The MIC was identified as the concentration corresponding to the dilution that exhibited the least inhibitory effect.

2.3.4. Effect of temperature, pH and enzymes on bacteriocin activity

In the heat tolerance assay, bacteriocin ZJQ2-1 was dissolved in phosphate-buffered saline (pH 7.2), following which the bacteriocin solution was incubated in a water bath at temperatures of 40°C, 60°C, 80°C and 100°C for 30 minutes. The assay also involved an autoclave process at 121°C for 30 minutes. In order to conduct the pH tolerance assay, bacteriocin ZJQ2-1 was suspended in PBS (pH 7.2), after which the pH was adjusted to 2, 3, 4, 5, 6, 7, 8, 9 and 10. Following a two-hour incubation period at room temperature, the pH was returned to 6.0 to negate the effects of acidity and alkalinity. In the assay of enzymatic degradation, the bacteriocin ZJQ2-1 was subjected to treatment with pepsin (1 mg/ mL, pH 2.0), trypsin (1 mg/ mL, pH 7.5), papain (1 mg/ mL, pH 7.5) and catalase (1 mg/ mL, pH 7.0) for a duration of two hours. Subsequent to this, the pH was adjusted to 6.0 (Peng, Xu, Fan, Qiao, Xie, Huang, et al., 2023). Corresponding controls were not subjected to enzyme, acid-base or heat treatments (20°C). Residual antibacterial activity was determined by the agar diffusion method. Each treatment assay was repeated three times.

2.4. The effect of P. pentosaceus JQ2-1 and bacteriocin ZJQ2-1 on B. cereus JN1L in fermented glutinous rice

The brewing process of fermented glutinous rice is as follows. First, 150 g of glutinous rice was washed twice in pure water and then soaked in pure water for 12 h. The soaked glutinous rice was placed in a steamer for steaming, and then 100 ml of sterile pure water was added to the steamed glutinous rice for cooling. To test the effect of P. pentosaceus JQ2-1 and its bacteriocin on spoilage bacteria during rice wine fermentation, Rhizopus arrhizus M5-1 (4×106 CFU/mL), Wickerhamomyces anomalus J5-1 (7×105 CFU/mL), Cyberlindnera fabianii J21-1 (3×105 CFU/mL), B. cereus JN1L (1×106 CFU/mL) were inoculated into cooled glutinous rice at 0.2% of the weight of glutinous rice, and P. pentosaceus JQ2-1 and bacteriocin were added as follows and fermented at 30°C for 48h. P. pentosaceus JQ2-1 and bacteriocin were added as follows: group A: without added bacteriocin ZJQ2-1 and P. pentosaceus JQ2-1(control group); group B: 5.5 log CFU/mL of P. pentosaceus JQ2-1 was added; group C: 32 µg/g of bacteriocin ZJQ2-1 was added; group D: 32 µg/g of nisin was added. Samples were taken from groups A to D at 12h intervals, and the quantity of B. cereus JN1L was calculated by dilution coating.

In order to study the effect of P. pentosaceus JQ2-1 and its bacteriocin on spoilage organism during the preservation of fermented glutinous rice, B. cereus JN1L (10⁶CFU/ mL) was inoculated into the fermented glutinous rice after pasteurization, and P. pentosaceus JQ2-1 and its bacteriocin were added as described above. Samples were taken every 3 days and the amount of B. cereus JN1L was counted by dilution coating.

2.5. Statistical analysis

The data analysis was performed using SPSS Statistics (Version 22, IBM Co., Somers, NY, USA). All data were the average representation of three experimental replicates, the results were shown as mean ± SD. The analysis of variance (ANOVA) test was used and p < 0.05 was considered significant.

2.6. Accession number

P. pentosaceus JQ2-1 was identified by 16S rRNA gene sequencing (GenBank accession number: PQ276996.1). The whole genome sequence of P. pentosaceus JQ2-1 has been uploaded to the GenBank database with accession number JBGTYH000000000.

3.1. Screening and identification of bacteriocin producing lactic acid bacteria

The 123 strains producing inhibition zones were obtained from 21 traditional sweet wine koji. Among them, strain JQ2-1 was cultivated in MRS agar plates at 37°C for 48 hours. Following this incubation period, round colonies of milky white and smooth consistency were formed (Fig. 1A). After excluding the effects of organic acids and hydrogen peroxide on the inhibitory activity, the supernatant of strain JQ2-1 showed the highest inhibitory activity against B. cereus JN1L. The bacteriostatic effect disappeared after treatment with trypsin and pepsin (Fig. 1B), indicating that strain JQ2-1 produced bacteriocin. As demonstrated in Fig. 1C, the phylogenetic tree of strain JQ2-1 indicates that the closest species to this strain is P. pentosaceus SL001.Therefore, strain JQ2-1 was identified as P. pentosaceus JQ2-1.

3.2. Purification and identification of bacteriocin

3.2.1. Analysis of whole genome sequencing results

In order to verify the probiotic properties of P. pentosaceus JQ2-1 at the genomic level, several probiotic-related genes were screened through genomic annotation. Genome-wide analysis of P. pentosaceus JQ2-1 was performed, revealing the presence of genes associated with temperature (htpX, hrcA, dnaK, dnaJ), bile tolerance (ppaC, cfa), and vitamin synthesis (fmnP, ribF, folA). The comprehensive list can be found in Table 1. Whole genome sequencing did not annotate bacteriocin-related genes, and in combination with the bacteriostatic effect of the strain, P. pentosaceus JQ2-1 may produce a novel bacteriocin.

Table 1

Probiotic genes in *P. pentosaceus* JQ2-1.
Gene	Function
Heat stress
htpX	zinc metalloprotease HtpX
hrcA	heat-inducible transcriptional repressor HrcA
dnaK	Chaperone protein DnaK
dnaJ	Chaperone protein DnaJ OS
Bile tolerance
ppaC	Manganese-dependent inorganic pyrophosphatase
cfa	cyclopropane-fatty-acyl-phospholipid synthase
Vitamin biosynthesis
fmnP	Riboflavin transporter FmnP
ribF	riboflavin biosynthesis protein RibF
folA	dihydrofolate reductase

3.2.2. Purification of bacteriocin

Carboxymethyl cellulose 52(CMC 52) chromatography was used to purify the bacteriocin separated from ethyl acetate. Three elution peaks were seen in the purification process of CMC 52, as shown in Fig. 2A. The agar diffusion method was used to assess the antibacterial activity of the eluted peak fractions and the results indicated that only A₃ exhibited antibacterial activity against B. cereus JN1L. Sephadex G-25 gel chromatography was used to purify the concentrated antibacterial active fraction (A₃). A total of two elution peaks were obtained (Fig. 2B), with B₂ showing antibacterial activity. The B₂ fraction was collected and purified using RP-HPLC, as shown in Fig. 2C, with a total of four elution peaks at 280 nm. The bacteriostatic activity of the eluted peaks was detected by agar diffusion method, and it was found that only the C₁ fraction showed bacteriostatic activity. The C₁ component was named bacteriocin ZJQ2-1. Data related to the purification process were summarized in Table 2.

Table 2

Purification results of bacteriocin
Purification stage	Volume (mL)	Total protein (mg)	Total activity (AU)	Specific activity (AU/mg)	Purification fold
CFS	500	1540.00	595854.92	386.92	1.00
Ethyl acetate	1	109.31	95751.30	875.96	2.26
CMC 52 cation chromatography	1	10.88	26839.38	2466.85	6.38
Sephadex G-25 gel chromatography	1	2.72	17253.89	6343.34	16.39
RP-HPLC	1	1.24	12849.74	10362.69	26.78

3.2.3. Amino acid sequence and molecular weight analysis of bacteriocin

The sequence of bacteriocin was analysed by HPLC-MS/MS, and the amino acid sequence of bacteriocin was measured as KIGLFGGAGVGKT. The amino acid sequence of the bacteriocin was analysed using Expasy, and the molecular weight of the bacteriocin was obtained as 1204.43 Da. Amino acid sequence comparison using Uniport and NCBI databases showed no similar matches, thus confirming that the purified bacteriocin was novel. Tricine SDS-PAGE results also showed a band less than 4100 Da (Fig. 3).

3.3. Characteristics of bacteriocin

3.3.1. Production kinetics of bacteriocin

Bacteriocin ZJQ2-1 as a metabolite was associated with the growth of strain P. pentosaceus JQ2-1. As shown in Fig. 4, bacteriocin ZJQ2-1 exhibited bacteriostatic properties from the 12-hour incubation time point onwards. The bacteriostatic capacity exhibited a gradual increase with the extension of the incubation time, reaching its maximum value at 24 hours, followed by a slight decrease. Consequently, 24 h was determined to be the optimal time point for the collection of bacteriocin. The OD₆₀₀ values showed that the growth curve of P. pentosaceus JQ2-1 was in the shape of "S", and P. pentosaceus JQ2-1 entered the logarithmic growth stage after 12 hours of incubation, and then entered the exponential growth stage, which was gradually stabilised after 24 hours. The pH curve showed that the pH of the culture medium of P. pentosaceus JQ2-1 only decreased slightly after 6 h of culture, sharply decreased between 6 and 15 h, and then slowly decreased, and the final pH value was about 3.8, indicating that it had a good acid-producing ability.

3.3.2. Antibacterial spectrum and MIC of bacteriocin ZJQ2-1

Bacteriocin ZJQ2-1 showed a good antibacterial spectrum (Table 3). In addition to the significant inhibitory effect of bacteriocin ZJQ2-1 on Gram-positive bacteria, such as Listeria monocytogenes ATCC 19116, Staphylococcus aureus CMCC 41002 and Bacillus subtilis FS SHOU, it also demonstrated a significant inhibitory effect on Salmonella CMCC15611, Escherichia coli ATCC 25922, Vibrio parahaemolyticus ATCC 33847 and other Gram-negative bacteria. From the table, it can be seen that bacteriocin ZJQ2-1 has no inhibitory effect on fungi such as Wickerhamomyces anomalus J5-1, Saccharomyces cerevisiae, Rhizopus orrhizus M5-1. This indicates that the addition of bacteriocin ZJQ2-1 to the fermentation process of fermented glutinous rice has no effect on the growth of fermentation microorganisms such as mould and yeasts. As shown in Fig. 5, the MIC of bacteriocin ZJQ2-1 was 16 µg/mL.

Table 3

Antimicrobial spectrum of bacteriocin ZJQ2-1
Indicator strains	Source	Medium and temperature (℃)	Zone of inhibition (mm)
G⁺
Listeria monocytogenes	ATCC 19116	LB, 37℃	18.8 ± 0.17
Staphylococcus aureus	CMCC 41002	LB, 37℃	19.3 ± 0.12
Bacillus subtilis	FS, SHOU	LB, 37℃	20.4 ± 0.15
Staphylococcus saprophyticus	FS, SHOU	LB, 37℃	18.9 ± 0.45
Bacillus cereus Z-3	FS, SHOU	LB, 37℃	20.9 ± 0.31
G⁻
Salmonella	CMCC 15611	LB, 37℃	17.4 ± 0.10
Escherichia coli	ATCC 25922	LB, 37℃	19.8 ± 0.38
Vibrio parahaemolyticus	ATCC 33847	LB, 37℃	17.0 ± 0.20
Fungus
Wickerhamomyces anomalus J5-1	FS, SHOU	YPD, 30℃	-
Cyberlindnera fabianii J21-1	FS, SHOU	PDA, 30℃	-
Saccharomyces cerevisiae	FS, SHOU	YPD, 30℃	-
Rhizopus arrhizus M5-1	FS, SHOU	YPD, 30℃	-

ATCC, American Type Culture Collection; CMCC, China Microbiological Collection Center; FS, SHOU, Food Science Laboratory of Shanghai Ocean University.

3.3.3. Stability of bacteriocin ZJQ2-1 after temperature, pH and enzymes

Figure 6 shows the effect of temperature, pH and protease on bacteriocin ZJQ2-1. From Fig. 6A, it can be seen that bacteriocin ZJQ2-1 has almost unchanged bacteriostatic activity at 20–60°C, and its bacteriostatic activity gradually decreases with increasing temperature. However, bacteriocin ZJQ2-1 still has good antibacterial activity after heat treatment at 100–121℃. In the pH tolerance test, the bacteriocin ZJQ2-1 showed high inhibitory activity in the pH range of 2.0–7.0, and the inhibitory activity tended to decrease with the increase of pH. At pH 8.0–10, the antibacterial activity of bacteriocin ZJQ2-1 decreased, but still retained high antibacterial activity (Fig. 6B). The results showed that the bacteriocin ZJQ2-1 could effectively inhibit bacteria in both acidic and alkaline foods. During enzyme treatment, pepsin, trypsin and Papain were able to deprive bacteriocin ZJQ2-1 of its inhibitory effect, while catalase had little effect on the inhibitory effect of bacteriocin ZJQ2-1 (Fig. 6C).

3.4. The effect of P. pentosaceus JQ2-1 and bacteriocin ZJQ2-1 on B. cereus JN1L in fermented glutinous rice

During the fermentation of the fermented glutinous rice, P. pentosaceus JQ2-1, bacteriocin ZJQ2-1 and nisin were inoculated in different ways. As shown in Fig. 7, the number of spoilage bacterium B. cereus JN1L gradually decreased to 1.08 log CFU/g with the growth of P. pentosaceus JQ2-1 (Fig. 7B). At 12 h of fermentation, the number of B. cereus JN1L did not decrease significantly, which may be due to the fact that P. pentosaceus JQ2-1 was in the exponential phase of growth and produced less inhibitory substances. After adding bacteriocin ZJQ2-1 ((Fig. 7C) and nisin ((Fig. 7D) for 48h, the number of B. cereus JN1L in the fermented glutinous rice decreased to 0.71 log CFU/g and 0.85log CFU/g, respectively.

When 5.5 log CFU/g P. pentosaceus JQ2-1 (Fig. 8B), 32 µg/g bacteriocin ZJQ2-1 (Fig. 8C) and 32 µg/g nisin (Fig. 8D) were added to the fermented glutinous rice during storage for 30 days, the levels of spoilage bacteria were reduced to 0.66 log CFU/g, 1.20 log CFU/g and 1.36 log CFU/g, respectively. The count of Bacillus cereus in the fermented glutinous rice that was inoculated with P. pentosaceus JQ2-1 and bacteriocin ZJQ2-1 decreased by about 80% and 64% of that in the initial stage, respectively. Therefore, the addition of P. pentosaceus JQ2-1 and its bacterin can effectively inhibit B. cereus JN1L in fermented glutinous rice.

Lactic acid bacteria produce organic acids, hydrogen peroxide, antimicrobial peptides, and other antimicrobial substances to control spoilage and disease-causing microorganisms in foods(Mokoena, Omatola, & Olaniran, 2021). Bacteriocin-producing LABs have attracted a lot of interest in recent years due to their potent inhibitory activity and safety. Some lactic acid bacteria that produce bacteriocin have been identified in cheese, fermented sausage and other foods.(Abrams, Barbosa, Albano, Silva, Gibbs, & Teixeira, 2011; Cavicchioli, Camargo, Todorov, & Nero, 2017). In this study, we obtained a bacteriocin-producing P. pentosaceus JQ2-1, isolated from the traditional sweet wine koji, whose bacteriocin strongly inhibited B. cereus JN1L.

Genetic annotation indicated that P. pentosaceus JQ2-1 has probiotic properties, and its application in rice wine fermentation can inhibit the growth of spoilage bacteria while exerting its probiotic effect. P. pentosaceus is rich in probiotic genes. Siriwan studied the whole genome of P. pentosaceus ENM104, and its functional annotation identified a number of genes associated with probiotic traits, including genes related to stress adaptation, bile tolerance vitamin biosynthesis, immunomodulation, and bacteriocin production(Kompramool, Singkhamanan, Pomwised, Chaichana, Suwannasin, Wonglapsuwan, et al., 2024). Eman isolated the marine pentosan P. pentosaceus E3 from the intestine of the shrimp, several genes consistent with probiotic microbial traits were estimated in the genome, including stress response, carbohydrate metabolism, and vitamin biosynthesis(Zaghloul & El Halfawy, 2024).

Bacteriocin ZJQ2-1 is a relatively small bacteriocin compared to other lactobacillus bacteriocins reported in previous studies, such as Pediocin SM-1 (5, 300 Da)(Anastasiadou, Papagianni, Filiousis, Ambrosiadis, & Koidis, 2008) and Pediocin 05–10 (6, 500 Da) (Huang, Luo, Zhai, Zhang, Yang, Tian, et al., 2009). Although typical bacteriocins have a molecular mass of > 2000 Da, low molecular weight bacteriocins have also been reported, such as plantaricin GZ1-27 (975 Da)(Du, Yang, Lu, Lu, Bie, Zhao, et al., 2018)、plantaricin JLA-9 (1044 Da)(Zhao, Han, Bie, Lu, Zhang, & Lv, 2016)、bifidocin A (1198.68 Da)(Liu, Ren, Song, Wang, & Sun, 2015) and L. salivarius SPW1(1221.074 Da)(Wayah & Philip, 2018). Small molecules of bacteriocins can play an antibacterial role not only by disrupting the integrity of bacterial cell membranes, but also by interacting with enzyme systems or cellular DNA, so their bactericidal efficiency tends to be better (Pei, et al., 2020; Zhao, Han, Bie, Lu, Zhang, & Lv, 2016).

The greater production of bacteriocin ZJQ2-1 during the stable phase of growth indicates that it is a secondary metabolite. Similar bacteriocin production by other lactic acid bacteria strains was also reported, such as bacteriocin KL-1(Komkhae, Kittaporn, Nualphan, & Doan Duy, 2015) and bacteriocin CG-9 (Cui, Pan, Xu, Li, Wang, Gong, et al., 2020). The decrease in antimicrobial activity of bacteriocins at the end of growth observed in this study may be due to the depletion of medium nutrients, triggering protein degradation to meet energy demand. Bacteriocin ZJQ2-1 can inhibit both gram-positive and Gram-negative bacteria. Similar observations were made for bacteriocin LB44 produced by P. pentosaceus LB44(Kaur & Tiwari, 2018), which showed inhibitory activity against both Gram-positive and Gram-negative bacteria. Therefore, bacteriocin ZJQ2-1 has a wide range of antibacterial applications. The MIC of bacteriocin ZJQ2-1 (16 µg/mL) was lower than enterocin CRL35 (57 µg/mL) (Caballero Gomez, Abriouel, Jose Grande, Perez Pulido, & Galvez, 2012), indicating higher potency. Bacteriostatic effect can be achieved by adding a small amount of bacteriocin ZJQ2-1 to food. Bacteriocin ZJQ2-1 has a high heat resistance, which is consistent with other bacteriocins such as lacritin MXJ32A (Lu, Yi, Dang, Dang, & Liu, 2014). The heat and pH-stable property of bacteriocin ZJQ2-1 is much superior to nisin, which is heat-sensitive and narrow pH range active(Fernandez-Perez, Saenz, Rojo-Bezares, Zarazaga, Rodriguez, Torres, et al., 2018).

The bacteriocin ZJQ2-1 not only had a bacteriostatic effect on the agar plate but also had a good bacteriostatic effect in the fermentation and preservation of fermented glutinous rice. Both P. pentosaceus JQ2-1 and its bacteriocin effectively inhibited the growth of B. cereus JN1L during fermented glutinous rice fermentation, and bacteriocin ZJQ2-1 was more effective than nisin in inhibiting the growth of B. cereus JN1L. The results of the bacteriostatic profile showed that bacteriocin ZJQ2-1 had no inhibitory effect on fermentation strains such as moulds and yeasts, and therefore bacteriocin ZJQ2-1 can be safely used in the fermentation of fermented glutinous rice. The direct addition of P. pentosaceus JQ2-1 was more effective than the direct addition of bacteriocin ZJQ2-1 during fermented glutinous rice preservation. This is because during the growth of lactic acid bacteria, not only bacteriocins but also antibacterial substances such as organic acids are produced(Zdolec, Hadziosmanovic, Kozacinski, Cvrtila, Filipovic, Skrivanko, et al., 2008). P. pentosaceus JQ2-1 and its bacteriocin have great potential in inhibiting spoilage bacteria in fermented glutinous rice.

This study screened a strain of P. pentosaceus JQ2-1 that produces bacteriocins from traditional sweet wine koji. After purification and identification, it produced a new type of bacteriocin ZJQ2-1, which has broad-spectrum antibacterial activity, good thermal stability, and pH stability. During the fermentation process, the addition of P. pentosaceus JQ2-1 and bactericin ZJQ2-1 could effectively inhibit the growth of B. cereus JN1L, but had no effect on the growth of the fermentation strains. The addition of P. pentosaceus JQ2-1 and bactericin ZJQ2-1 can inhibit the growth of spoilage bacteria and prolong the shelf life of fermented glutinous rice, which is of great significance to the fermented glutinous rice production industry.

Ethics approval and consent to participate

This paper does not involve human and animal experiments, so it does not need ethical approval and informed consent.

Consent for publication

All the authors agree to publish the paper.

Competing interests

The authors declare that they have no competing interests

Funding

This study was supported by 2025 Karamay Science and Technology Plan Project-Technology Innovation Guidance Plan Project (2025CA0021)

Authors' contributions

Huojian Zheng: Writing-original draft; Writing-review & editing, Data curation. Yongqi Yang: Methodology, Investigation, Data curation. Cancan Qi: Methodology, Investigation, Data curation. Qiuxiang Cheng and qin Wang: Methodology, Investigation. Liping Wang: Resources, Project administration, Funding acquisition.

Acknowledgements

Thanks to Professor Liping Wang for his valuable revision opinions. And then, thanks to our colleagues Yongqi Yang and Cancan Qi for their help in the paper.

Availability of data and materials

Data and information available

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Bacteriocin ZJQ2-1 produced by Pediococcus pentosaceus from traditional sweet wine koji: purification, characterization, and application in fermented glutinous rice

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Screening and identification of bacteriocin-producing lactic acid bacteria

2.1.1. Isolation of bacteriocin-producing lactic acid bacteria

2.1.2. Identification of strain

2.2. Purification and identification of bacteriocin

2.2.1. Whole genome sequencing and annotation

2.2.2. Purification of bacteriocin

2.2.3. HPLC-MS/MS analysis of bacteriocin ZJQ2-1

2.2.4. Molecular mass analysis of bacteriocin

2.3. Characteristics of bacteriocin

2.3.1. Production kinetics of bacteriocin

2.3.2. Antibacterial spectrum of bacteriocin ZJQ2-1

2.3.3. Determination of minimum inhibitory concentration

2.3.4. Effect of temperature, pH and enzymes on bacteriocin activity

2.5. Statistical analysis

2.6. Accession number

3. Results

3.1. Screening and identification of bacteriocin producing lactic acid bacteria

3.2. Purification and identification of bacteriocin

3.2.1. Analysis of whole genome sequencing results

3.2.2. Purification of bacteriocin

3.2.3. Amino acid sequence and molecular weight analysis of bacteriocin

3.3. Characteristics of bacteriocin

3.3.1. Production kinetics of bacteriocin

3.3.2. Antibacterial spectrum and MIC of bacteriocin ZJQ2-1

3.3.3. Stability of bacteriocin ZJQ2-1 after temperature, pH and enzymes

4. Discussion

5. Conclusion

Declarations

Ethics approval and consent to participate

Funding

Authors' contributions

Acknowledgements

Availability of data and materials

References

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