Inhibition of Lactobacillus fermentum SHY10 on the white membrane production of soaked pickled radish

Abstract The formation of white bio‐membrane (shenghua) on the surface of pickle leads to uneatable and spoiled products, which has been the key problem restricting the development of Sichuan pickle industry. In this study, the 17 microorganisms in the white membrane of pickled radish were screened and identified, of which Candida parapsilosis was the main strain causing ”shenghua“. The membrane‐forming ability of Candida parapsilosis was determined by crystal violet staining to explore its adaptability to the fermentation environment concerning temperature and oxygen. It was found that Candida parapsilosis had the strongest membrane‐forming capacity under the aerobic condition at 37°C, with the highest OD595 nm value reached to 3.473 ± 0.07 at 72 h post inoculation. This research identified Lactobacillus fermentum SHY10 to be the inhibitor of the membrane production of Candida parapsilosis via the Oxford cup method on a Petri dish, and via co‐inoculation with Candida parapsilosis in pickles. Furthermore, this study specified that the cell‐free supernatant (CFS) of L. fermentum SHY10 had the most significant inhibitory effects and likely to result from protein substances in the CFS. Proteases treated CFS had significantly reduced inhibitory effects against membrane formation, which confirmed that the active component was protein substances. Overall, this study identified a functional LAB strain with significant inhibitory effects against the white membrane formation in pickles, which provide a safe and consumer‐friendly solution for the membrane problem in the fermented vegetable industry.

In commercial production, high salinity pickling was used to control white membrane formation to some extent. However, high salinity pickling has apparent disadvantages as it limits the growth of desirable probiotics and prevents the formation of unique flavors. In this study, strains causing white bio-membrane in pickles were isolated and identified using Oxford cup and whole-genome sequencing. Lactic acid bacteria (LABs) have been used as safe bio-preservatives for various food products, including dairy, meat, aquatic products, fruits, and vegetables (Zhang et al., 2017).
Previous research has demonstrated the effectiveness of LABs controlling the growth of undesirable bacteria in fermented products.
For example, Lactobacillus plantarum AJS2-4 screened from Jixi sour cowpea, Anhui Province, China was reported to inhibit quorum sensing and bio-membrane formation of Aeromonas hydrophila in sour cowpea . Anran (2021) found that bacteriocin of Streptococcus lactis suppressed the growth of Listeria monocytogenes on instant ham and lettuce. Zhao et al. (2019) identified Pichia guilliermondii Y35-1 with significant antibacterial effects on Colletotrichum gloeosporioides after harvest. Overall, LABs have good potential as a natural biological preservative in the field of food preservation.
In this study, the bacteria and fungi causing white membrane formation on the surface of pickles were isolated and identified via molecular biological identification techniques. This study also explored the adaptability of membrane-producing strains to the fermentation conditions, namely, temperature and presence of oxygen.
Importantly, natural protection against white membrane formation in pickles was investigated, and Lactobacillus fermentum SHY10 was screened and identified to be effective in inhibiting the growth of membrane-forming yeast. The inhibitory efficacy was confirmed both on the plate and in the making of fresh pickles. The identification and verification of Lactobacillus fermentum SHY10 being an effective antimembrane bio-presentative are meaningful to the pickle industry. It is a safe and consumer-friendly approach to produce high-quality pickles. This study confirmed that effective components are protein substances, while the mechanism of the protective effects of L. plantarum SHY 10 against "shenghua" remains unclear and requires further exploration.

| Materials
Fresh raw white radish was washed by DI water, peeled, and chopped into small pieces (approximate 3 × 5 × 5 cm). Chopped radish were transferred to bottles with salty water to achieve 6%-8% salinity. A headspace is left on top of the bottle. The bottles were then sealed and left in a cool and shady place to ferment until the "shenghua" was observed. "Shenghua" pickles were collected from 10 samples of pickled radish from five Chongqing households. The surface of the pickles showed fragmented white bio-membranes, and the pickles had turbid water and pungent acidic odor. During sampling, the white flowers on the surface of the jar were carefully picked with a glass rod and transferred to a centrifuge tube as sample 1, and 5-ml pickled vegetable water was taken and transferred to a centrifuge tube as sample 2. No radish was collected. Samples 1 and 2 were brought back to the laboratory in an ice cube box and stored at 4°C for further use. All supplies during sampling are sterilized in advance.

| Isolation and identification of membraneforming microbes
A small piece of the white membrane was picked by an inoculation ring, mixed in 5-ml normal saline, then diluted to the appropriate gradient. The 1-ml pickled water with "shenghua" in sample 2 was taken by a liquid transfer gun, mixed in 9-ml normal saline, and then diluted to the appropriate gradient. The 100 μl of each culture was spread on MRS medium and YPD medium. Each gradient has triplicates, and all samples were cultured in a 37°C constant temperature incubator (Shanghai Yuejin Medical Device Co., Ltd.) for 48 h. Single colonies were picked and repeatedly streaked and purified until the pure culture was obtained. The colony morphology was observed under a microscope. The single pure colony was selected and inoculated in MRS liquid medium and YPD liquid medium, respectively. Both cultures were incubated at 37°C for 24 h and placed at 4°C for further use.

| Bacterial 16S rDNA and yeast 18S rDNA amplification and sequencing identification
The lactic acid bacteria genome was extracted using the Solebao bacterial genomic DNA extraction kit (Beijing solarbio Science and Technology Co., Ltd.). The 16S rDNA fragment was amplified using the bacterial 16S rDNA universal primer 27f as the upstream primer, 1492r as the downstream primer, and genomic DNA as the template. PCR reaction procedures were described below: Pre-denaturation at 94°C for 3 min for 30 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min), extension at 72°C for 5 min.
Yeast genome was extracted by Solebo Yeast Genome DNA Extraction Kit (Beijing solarbio Science and Technology Co., Ltd.).

Practical Application
This study identified a functional lactic acid bacteria strain with significant protective effects against white membrane formation in pickles. Identifying and verifying the strain are meaningful to the pickle industry that white film has been a costly problem for the fermented vegetable industry. This study provides a safe and consumer-friendly approach to produce high-quality pickles.
Yeast 18S rDNA was amplified with universal primers ITS1 and ITS4, and genomic DNA was used as a template. PCR reaction program was set up as below: Pre-denatured at 95°C for 5 min, 25 amplification cycles (denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min), and elongation at 72°C for 10 min.
The PCR products were then sent to Majorbio Bio-Pharm Technology Co., Ltd for sequencing. The total reaction volume of the system is 20 μl. Add 2.0 μl 10 × Ex Taq buffer, 1.6 μl 2.5 mM dNTP, 0.5 μl template DNA, One pair of primers each 1 μl, 0.2 μl 5 U Ex Taq, add ddH2O to 20 μl. The obtained sequences were BLAST (https:// www.ncbi.nlm.nih.gov/) to perform the nucleic acid data comparison and gene identification.

| Isolation and purification of white membraneforming strains
The isolated bacteria and yeasts were inoculated into MRS and YPD liquid medium, respectively, and cultured at 37°C. The membrane production on the surface of the microbial liquid was observed and recorded. The microbial isolates with good membrane production were selected, isolated, and cultured at 37°C for 18 h in liquid culture and placed at 4°C until further analyses. The 100μl seed solution was inoculated in the 10-ml sterile liquid culture medium and cultured at 37°C. The absorbance was measured at 600 nm by Ultraviolet-Visible spectrophotometer (Japan Shimadzu Company) every 2 h. The growth curve was drawn with time as abscissa and OD value as ordinate (Cui et al., 2018).

| Validation of membrane-producing ability of test strains
To validate the membrane-producing ability of test strains, the strain inoculum was added in the pickle-making process and the pickle samples were observed for membrane (bio-membrane) formation. The preparation of pickle samples can be summarized as below: cleaning fresh radish → drying → cutting → weighing → vegetable pieces was immersed in 6% (v/v) saltwater (brine) → samples inoculated with the test strain. The strain was cultured to 10 6 CFU/ ml and inoculated into pickles to reach 0.4 % (v/v) concentration of the pickle brine→ fermentation. A control sample following the same procedures was prepared, except the control was not inoculated with the test strain.

| Adaptability of membrane-producing strains to oxygen and temperature
To explore the membrane-forming conditions concerning oxygen, the selected membrane-producing strains were inoculated into a liquid culture medium and cultured at both aerobic and anaerobic conditions. The experiment was performed using the dilution spread plate method as described by Du et al. (2016) with slight modification. The membrane formation process was observed, and the colonies were counted at 24, 48, and 72 h, respectively.
Oxygen level and temperature are the two conditions that have not been fully understood and requires further control in the current Chinese pickle processing. To explore the membrane-forming condition concerning temperature, the selected strains were inoculated into liquid culture medium at 10, 25, and 37°C, respectively, at aerobic conditions using an air penetrable bottle cap. To analyze the effects of oxygen on membrane forming, the selected strains were inoculated into liquid culture medium. One group was air-tight capped and the other one was not fully sealed and capped with an air penetration cap. The plates were counted and checked for membrane formation at 24, 48, and 72 h, respectively. Crystal violet staining method was used to detect the membrane formation ability.

| Analysis of the effects of Lactobacillus fermentum SHY10 supernatant, thallus, and extracellular metabolites on Candida parapsilosis B7
Lactobacillus fermentum SHY10 was inoculated and cultured in MRS liquid medium at 2% (v/v) at 37°C for 48 h. The bacteria culture was then centrifuged at 4°C at 3996 g for 15 min, and the supernatant was filtered to obtain cell-free supernatants (CFS). The CFS was transferred to the culture plates, frozen at -80°C, and vacuum freeze-dried (Beijing Songyuan Huaxing Technology Co., Ltd.) until further analyses. When used, 1 ml of sterile distilled water was added to the culture plate to obtain a concentrated supernatant . The cells were then collected and divided into two groups. One group was washed twice with sterile water while another group was untreated to obtain the washed and unwashed Lactobacillus plantarum, respectively (Liu, 2019).

| Effect of proteases on CFS of Lactobacillus fermentum SHY10
Four aseptic supernatants of Lactobacillus plantarum were prepared and were adjusted to the optimal pH of pepsin, papain, trypsin, and protease K, individually. Each protease was added to an extracted solution adjusted to the protease's corresponding optimal pH to reach the final mass concentration of 1.0 mg/ml. The pH value was then adjusted to the original value of the CFS. The activity of untreated sterile supernatant was determined by Oxford cup method .
The culture of Candida parapsilosis B7 suspension was diluted to approximate 10 6 CFU/ml using MRS broth. The inoculum of Candida parapsilosis B7 was mixed into YPD medium (1% agar) to reach 0.5% (v/v) concentration. The mixture (20 ml) was poured into a YPD plate with a sterilized Oxford cup placed up still in the plate. After cooling, the Oxford cups were taken out carefully to form round holes.
Various volumes of L. plantarum CFS, unwashed L. plantarum suspension, and washed L. plantarum suspension were added to the small round holes. MRS medium was used as the control group. Plates were incubated at 37°C for 24 h, and the opaque circle diameters around the well were measured using a ruler. The experiment was performed in triplicate, and the data were recorded at mean ± standard deviation.

| Statistical analysis
Excel 2010 software was used to analyze the data. Experiments were performed in triplicate, and results were expressed as the mean value ± standard deviation. GraphPad Prism 7.0 was used to process graphs.

| Isolation and identification of "shenghua" (white membrane) microorganisms
Bacterial sequences were compared with known sequences by the BLAST in the DNA database of NCBI online tool. The results of BLAST identification and homology of strains are shown in Table 1.
A total of 17 isolates were isolated in this experiment, which were identified as Lactobacillus plantarum (5 strains (Mao et al., 2018;Rao et al., 2018). Enterococcus faecium can shorten fermentation time, control nitrite content, and inhibit the growth of spoilage microorganisms (Lu et al., 2017). Bacillus are the main spoilage bacteria in pickles. Bacillus altitudinis exists in soil, introduced in the final product via unsterilized raw materials .
Klebsiella is related to the bloating bags of pickle products (Ao et al., 2019;Cai et al., 2017). Candida widely exists in the environment and can be found on vegetables and soil surfaces that could cause fer-

| Screening and validation of membraneproducing microorganisms
3.2.1 | Screening and growth curve of membraneproducing strains The purified strains were inoculated in a liquid medium, and a blank medium was used as a control. The cultures were monitored for bio-membrane formation, and the strains capable of forming biomembrane were selected. As shown in Figure 1

| Verification of membrane-producing strains
To verify whether Candida parapsilosis has the membrane-producing ability that caused the "shenghua" of pickles, Candida parapsilosis was inoculated in fresh pickles, and the pickles without Candida parapsilosis inoculation were the control. As seen in Figure 3  were previously reported to cause white membrane formation on the surface of fermented vegetables (Lu et al., 2019;Zhang et al., 2016). In this study, Candida parapsilosis was found to be the main strain causing pickle "shenghua".

| Antibacterial test of Lactobacillus fermentum SHY10
To evaluate the inhibitory effects of Lactobacillus fermentum SHY10

| Analysis of inhibitory effects of Lactobacillus fermentum SHY10 on Candida parapsilosis B7
To investigate the antimembrane forming substances of Lactobacillus fermentum SHY10 CFS, the thallus and extracellular metabolites of thallus of Lactobacillus fermentum SHY10 were extracted and inoculated with Candida parapsilosis B7 using the Oxford cup method as described in 2.8. After 48 h of incubation, the diameters of inhibition zones on YPD plate were measured with a ruler (Table 5). The diameters of inhibition zone increased as the amount of CFS increased, but all volumes showed significant inhibition effects compared to the control (p < .05). According to Figure

| Effect of proteases on CFS of Lactobacillus fermentum SHY10
The effective substances in L. plantarum were previously reported to be organic acids and bacteriocin (Wang et al., 2020). Bacteriocins are proteins or antimicrobial peptides that are synthesized by bacterial ribosomes that form channels on the cell membrane of target bacteria or directly inhibit specific functions of target bacterial cells and efficiently exert bactericidal effects (Cleveland et al., 2001). To further investigate if (part of) the antimicrobial components belong to the bacteriocin category and the type(s) of proteins, the CFS of L. plantarum was digested by four commonly used proteases against bacteriocin produced by Lactobacillus, namely pepsin, papain, trypsin, and proteinase K . The digested materials were evaluated for the inhibitory efficacies to explore the type(s) of active components presented in CFS. The supernatant of L. plantarum was treated with each protease, separately. Each protease was added at its optimal pH to ensure the maximal efficacy of the enzyme. The proteases treated CFS were used to perform the inhibition zone test against Candida parapsilosis, individually. Interestingly, the inhibitory effects were no longer perceived or significantly weakened compared to untreated CFS (control) (Figure 6).
Previous work in our laboratory screened and purified the strain of Lactobacillus fermentum SHY10 from pickles. Lactobacillus fermentum SHY10 was found to be effective against Staphylococcus aureus due to peptides secreted by the bacteria .
Specifically, three antibacterial peptides (ABPs) produced by Lactobacillus fermentum SHY10 were identified. Among the ABPs, the NQGPLGNAHR peptide showed the most effective antibacterial activity, which may be associated with its interaction with dihydrofolate reductase (DHFR) and DNA gyrase of membrane-forming microorganisms .
According to Figure 6, the digestion by proteases led to no inhib-  (Kong et al., 2021).
Overall, in this study, the effects of Lactobacillus fermentum SHY10 against the membrane-forming yeast were explored and confirmed, and the functional chemical substances were investigated using a protease digestion experiment. The study further confirmed that bacteriocin-like substances secreted by Lactobacillus fermentum SHY10 played a key role in preventing the formation of white membrane in pickles. Further researches are required to specify the type of bacteriocin and understand the exact mechanism of inhibition.

| CON CLUS ION
In this study, a total of 17 isolates of microorganisms were isolated from "shenghua" pickled radish. The results of molecular biology F I G U R E 4 Appearance of pickles inoculated with membraneproducing yeast (a) and pickles inoculated with both membraneproducing yeast and Lactobacillus fermentum SHY10 (b) Diameter of the transparent circle of suppression circle (cm) 0* 0.47 ± 0.03* 0.82 ± 0.01* 1.14 ± 0.01* 2.32 ± 0.02* 2.61 ± 0.03* *Indicates significant differences among all date points.
identification and back inoculation test showed that Candida parapsilosis was the main microorganism causing "shenghua" of pickled radish.
This study also identified that the optimal conditions for Candida parapsilosis to produce bio-membrane were under aerobic condition at 37°C.
Lactobacillus fermentum SHY10 was explored for the antimembrane formation caused by Candida parapsilosis in pickles. This study showed that the effects of inhibition mainly resulted from the CFS supernatant of Lactobacillus fermentum SHY10. To further understand the functional substances of membrane inhibition, the CFS was treated with different proteases then tested for inhibitory efficacy. The results showed no inhibitory effect or the inhibitory effect was significantly weakened after the protease treatment, indicating that the active component(s) of its supernatant were likely to be bacteriocin-like substances.
In summary, the CFS of Lactobacillus fermentum SHY10 significantly inhibited the growth of Candida parapsilosis in pickles. As a microbe naturally exist in fermented products, the protection from Lactobacillus fermentum SHY10 is a safe treatment to prohibit the growth of white membrane in pickle products. Further studies should focus on the isolation and purification of the functional peptides secreted by Lactobacillus fermentum SHY10. Researches should also be performed to understand the mechanism behind the inhibitory effects in order to provide theoretical support for the study of the inhibition of white membrane formation in pickles.