In vitro probiotic screening and evaluation of space‐induced mutant Lactobacillus plantarum

Abstract Space mutation is an efficient tool in microbial breeding. The aim of the present study was to screen out space‐induced mutants of Lactobacillus plantarum with potent probiotic properties. The wild‐type Lactobacillus plantarum GS18 was subjected to 31 days and 18.5 hr of space flight, in which 13 isolates were selected for analysis. These mutants were assayed for milk fermentation performance, low pH resistance, bile salt tolerance, hydrophobicity, and antimicrobial activity. The 16S rDNA sequencing was applied to identify the stain and compare it with the wild type. Results showed that the isolate L. plantarum SS18–50 had the strongest probiotic properties with no mutation in 16S rRNA sequence compared to the wild type. Specifically, L. plantarum SS18–50 had good milk fermentation performance. The viscosity of fermented milk, acid tolerance, and bile salt tolerance were increased by approximately 10%, 8%, and 9%, respectively (p < .05). The antibacterial activity (Escherichia Coli, Salmonella Typhimurium, and Listeria Monocytogenes) was also increased significantly compared to the wild type (p < .05). This study indicates that L. plantarum SS18–50 has the great potential to serve as a probiotic for dairy products.

Lactobacillus plantarum is a kind of lactic acid bacteria, anaerobic or facultative anaerobic. L. plantarum has been reported to have many health beneficial functions (Vries, Vaughan, Kleerebezem, & Vos, 2006). Since space mutagenesis is an efficient strategy for microbial breeding by which we have successfully used in L. reuteri screening, in the current study we intended to screen L. plantarum -a commonly used probiotic in dairy industry, with enhanced probiotic characteristics by space flight mutation.

Lactobacillus plantarum GS18 provided by Fullarton Biological
Engineering Technology Co., Ltd (Beijing, China) was anaerobically grown in MRS medium at 37°C and used as the wild type sent for space mutagenesis. The pathogens including Escherichia Coli, Staphylococcus Aureus, Salmonella Typhimurium, and Listeria Monocytogenes (ATCC) were provided by Jilin Entry-Exit Inspection and cultured in TSB medium at 37°C with shaking at 100 rpm.

| Space mutagenesis
Lactobacillus plantarum GS18 was inoculated into semisolid MRS medium containing 0.5% agar in plastic tubes and cultured in a stationary incubator at 37°C for 48 hr under anaerobic condition. One tube was sent to outer space in a special biological bag of the re-entry capsule by the China's Shenzhou-11 spacecraft at 07:30am Beijing time on October 17, 2016, and traveled at 393km height for 31 days and 18.5 hr before landing. The other tubes were left on the ground and kept at 4°C as a control in the analysis.

| Performance in skim milk fermentation
The bacteria were cultured into MRS broth and streaked on MRS agar plates. Eighty-eight colonies were randomly selected and cultured for over five generations to assure incapability of reverse mutation before use in this experiment. Each of them (0.5ml) was inoculated in a tube containing 5 ml 12% skim milk and cultured at 37°C. The milk coagulation time and the pH value were recorded. Performance with coagulation time less than 6h and pH less than 4.5 was considered excellent.
Sixteen isolates were selected based on their coagulation time and pH of the fermented milk. These isolates were put into skim milk medium and cultured at 37 o C. After curding, the viscosity was measured by a viscometer (Brookfield LVDV Ⅲ U) under room temperature.

| Resistance to low pH and bile salt
The pellet of culture was suspended in either PBS buffer (pH 2.5) and then incubated in an orbital shaker (100 rpm) at 37°C for 3 hr or in PBS buffer (pH 8.0) containing 0.5%(w/v) bile salts (Yuanye Biotechnology, Shanghai, China) which was then incubated in an orbital shaker (100 rpm) at 37°C for 4 hr. The total viable counts were determined by counting colonies on the MRS agar after cultivation at 37°C for 48 hr.
Resistance index (RI) was calculated as follows: N 0 = initial cell number, N f = final cell number.

| Cell surface hydrophobicity
The bacterial solution in PBS buffer was adjusted to 0.8-1.0 (A 0 ) at 600 nm . The suspension was added with xylene at 3:1 (v/v) and then fully shook for 2 min. After 15 min incubation at ambient temperature, the aqueous phase was carefully sampled and measured at 600 nm using a UV-2550 spectrophotometer (Shimadzu, Japan). The hydrophobicity (H %) was calculated as follows: A 0 : initial absorbance; A absorbance after mixing with xylene.
The isolates with H % ≥50 were considered to be hydrophobic.

| Automatic aggregation
The bacteria in PBS (pH7.2) (OD600 = 0.7-1.0) were cultured in a constant temperature incubator at 37℃. Take 2 ml solution cultured for 6 hr and 12 hr respectively to determine the absorbance value at 600 nm. The automatic aggregation ability was calculated as follows:

| Antibacterial activity
The tested bacteria were cultured in MRS for after 24 hr incubation and then centrifuged at 6,000 g at 4°C for 10 min. The supernatant was adjusted to pH = 6.5 with 1M NaOH and then filtered by a 0.22 μm filter. The filtrate (100 μl) containing tested bacteria was added with 100 μl of 10 5 CFU/mL specific pathogen (E. coli, S. aureus, S. typhimurium, and L. monocytogenes) in a 96-well microtiter plate and incubated for 24 hr at 37°C in an orbital shaker (100 rpm) before measurement at 600nm. Antibacterial activity was calculated as follows: OD control: absorbance without pathogens; OD treatment: absorbance cocultured with specific pathogen.

| Statistical analysis
All experiments were performed in replicate or triplicate. The data were presented as mean ± standard deviation (SD). The difference was evaluated by Students' t test using IBM SPSS Statistics 19 at significance level at p-value < .05.

| Skim milk fermentation performance of eightyeight mutant isolates
Based on milk fermentation assay, 16 isolates of bacteria were selected for further examination, as they showed shorter coagulation time (≤ 6 hr) for milk and lower pH (≤4.5) compared with the wild type (Table 1).

| pH and bile salt tolerance of sixteen mutant isolates showing desirable skim milk fermentation performance
Probiotics have to go through the harsh environment of acid and bile to approach the host gut microbiome (Bezkorovainy & Anatoly, 2001). In general, all selected isolates showed good tolerance to low pH and bile salt compared with the wild type. It is hard to obtain mutants in good tolerance to pH and bile salt by random mutagenesis using traditional methods, whereas space mutagenesis is efficient to generate more beneficial mutations attributable to modification of metabolic capability (Zhang, Fang, & Liu, 2015). After 3 hr of low pH exposure, 11 stains were more resistant to low pH than the wild type. For bile salt resistant, SS18-21 and SS18-50 showed more bile salt tolerant than the wild type, of which only SS18-50 showed both resistance to low pH and bile salt (Table 2).

| Hydrophobicity and self-aggregation activity of sixteen mutant isolates showing desirable skim milk fermentation performance
The hydrophobicity and self-aggregation ability of bacteria are two features that affect the adhesion of bacteria to host tissues (Pascual et al., 2008). The high hydrophobicity of cell surface contributes to the colonization of bacteria on the surface of intestinal mucosa and promotes the adhesion of bacteria to intestinal epithelial cells. The hydrophobicity of 16 mutant isolates to xylene was determined, of which 12 isolates had similar hydrophobicity, and 4 isolates (SS18-28, SS18-50, SS18-51, and SS18-67) had significantly higher hydrophobicity than the standard strain GS18 (p < .05). These mutants were considered hydrophobic with H% ranging from 7% to 14% (Figure 2). Adherence to intestinal epithelial cells is an integral probiotic characteristic for transient colonization of probiotics needs the bacteria to adhere to intestinal epithelial cells which is determined by their hydrophobicity (Zhang et al., 2015).

TA B L E 1 Aggregation time and pH of milk fermented by different bacteria
The self-aggregation ability of bacteria is another one of the indicators that determine the adhesion of bacteria to host tissues. All mutant isolates showed good self-aggregation ability in both 6 hr and 12 hr. Typically, three isolates (SS18-37,  were significantly different from the standard strain GS18 in 12h self-aggregation (p < .05) (Figure 3).

| Antibacterial activity of sixteen mutant isolates showing desirable skim milk fermentation performance
Antimicrobial activity against harmful gut pathogens is considered one of the important characteristics of probiotics (Servin & Coconnier, 2003). The supernatants of MRS of most mutant isolates showed powerful antibacterial activity against 4 tested pathogens. Probiotics in the process of culture will produce bacteriocin, which has inhibitory effect on pathogenic bacteria in food. Additionally, lactic acid bacteria like L. plantarum can also F I G U R E 1 Viscosity of the fermented milk. The viscosity of most bacteria was similar while 6 mutant isolates were significantly higher than that of the wild-type fermented milk (p < .05)

TA B L E 2 Resistance to pH and bile salt
No.

F I G U R E 2
Hydrophobicity of the space mutant isolates. The hydrophobicity of 4 mutant isolates (SS18-28, SS18-50, SS18-51, and SS18-67) had significantly higher hydrophobicity than the wild type GS18 (p < .05) F I G U R E 3 Self-aggregation ability of the space mutant isolates. All tested bacteria showed good self-aggregation ability in both 6 hr and 12 hr, of which 3 isolates (SS18-37, SS18-67, and SS18-51) had significantly higher self-aggregation ability than the standard isolate GS18 (p < .05) generate acid microenvironment which can inhibit pathogens (Güven & Benlikaya, 2010). The bacteriostatic effect of 17 isolates was between 20%-30%. Compared with the wild-type GS18, two isolates (SS18-35 and SS18-50) showed significantly higher antibacterial ability (Table 3). Our research showed that space mutagenesis can improve antimicrobial activity of L. plantarum. These findings indicated that the outer space environment may modify some secondary metabolites that inhibit the growth of typical pathogens.

| 16S rDNA sequence comparison of the wild type and the mutant isolate SS18-50
Based on the 16S rDNA sequence, the SS18-50 was identified as the species L. plantarum. The 16S rDNA sequence of the wild type and SS18-50 were the same (data not shown). Space mutagenesis did not induce variation of 16S rDNA sequence in our experiment, indicating that 16S rDNA is resistant to space mutagenesis. The enhanced probiotic property was likely attributed to the change of the metabolic pathway.

| CON CLUS ION
Space mutagenesis is found to be an effective way for improving probiotic characteristics. The mutant L. plantarum SS18-50 was found to possess stronger probiotic characteristics with no variation in 16S rDNA sequence compared to the wild type. L. plantarum SS18-50 is a potential probiotic that can be used in fermented food industry.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.