Antibacterial and Antifungal Activity of Lactobacillus Plantarum Isolated from Green Tea

Background: Balanced skin microora is crucial for maintaining healthy normal skin function; however, changes in skin microora are associated with skin diseases such as acne vulgaris, dandruff, and candidiasis. Lactobacilli are probiotics that possess antibacterial activity against pathogenic bacteria. In the present study, we assessed the potential antagonistic activity of Lactobacillus plantarum APsulloc 331261 and 331266, which were isolated from green tea (Camellia sinensis), in inhibiting the growth of bacteria (Cutibacterium acnes) and fungi (Candia albicans, Malassezia globosa, and Malassezia restricta) related to skin diseases. Results: The antibiotic susceptibility of C. acnes, C. albicans, M. globosa, and M. restricta was tested using agar overlay and co-culture transwell methods to determine the antibacterial and antifungal activity of L. plantarum APsulloc 331261 and 331266. Results of the agar overlay method revealed that the two L. plantarum strains, APsulloc 331261 and 331266, inhibited the growth of C. acnes and C. albicans, respectively (zone diameters of inhibition: 20.0 ± 2.0 to 27.0 ± 3.6 mm; R values: 4.3 ± 1.8 to 5.5 ± 1.7). Moreover, the conditioned media (cell-free culture supernatants) derived from L. plantarum APsulloc 331261 and 331266 inhibited the growth of C. acnes, C. albicans, M. globosa, and M. restricta. Conclusions: These results suggest that L. plantarum APsulloc 331261 and 331266 could be used as candidate probiotics to control the balance of skin microora.


Background
Probiotics are gaining immense attention in scienti c research and are widely consumed worldwide due to their bene cial effects on health [1,2]. Lactic acid bacteria (LAB) produce lactic acid as a major product through carbohydrate fermentation, and Lactobacillus forms the major part of this group, as it is abundantly present in the intestines of vertebrates [3]. Lactobacillus plantarum is considered one of the most widely used probiotics in the food and pharmaceutical industries [4]. It undergoes unique metabolic processes to produce numerous vitamins and host immunomodulatory substances [5,6]. L. plantarum produces various antibacterial compounds such as hydrogen peroxide, acids, and bacteriocin [7].
Bacteriocins are peptides that inhibit or kill the bacterial strains; however, in general, they are harmless to the human body and surrounding environment [8,9]. Bacteriocins are less toxic than chemically synthesised antibiotics because they are resistant to proteases in the gastrointestinal system [10,11].
The skin possesses diverse ora, which act as a defence mechanism against pathogenic invasions by producing protein complex antibiotics [12,13]. Disruptions in the balance between the host and microorganism can lead to skin diseases or infections [13]. Cutibacterium acnes, a gram-positive and aerotolerant bacterium, is the primary cause of acne [14]. The onset of acne occurs in the sebaceous gland of the hair follicle, and various factors contribute to the secretion of the sebaceous glands and cause abnormal keratinisation in the hair follicles, resulting in clogging of the hair follicles and acne symptoms. Furthermore, it triggers in ammation with redness and oedema, further aggravating acne symptoms [15,16]. Malassezia, the main cause of dandruff, is a normal skin ora found in approximately 75-98% of healthy adults [17]. In Korea, Malassezia restricta and Malassezia globosa are commonly linked to dandruff [18]. Although Malassezia does not produce its own essential fatty acids for survival, it comprises eight lipases with three types of phospholipases [19]. The sebum produced in the scalp is decomposed by lipase to create oleic acid. Oleic acid triggers dandruff by rapidly dividing the skin cells in humans who are more sensitive than normal individuals, after passing the outer layers of the skin [20]. In candidiasis, Candida infects the skin, ngernails, mucous membranes, and other parts. Candida is a commensal fungus normally present in humans; however, when the immune system of the body is compromised or the epidermis is damaged by trauma, it causes infections [21][22][23]. Candida albicans causes 70-80% of all Candida infections, and tinea pedis is a candidiasis that occurs on the ngertips [24]. C. albicans is also associated with dandruff [25].
To treat skin diseases occurring due to imbalanced skin microbiome, antibiotics and antifungals are usually administered either orally or topically to suppress the bacterial and fungal growth; however, these may have adverse effects, such as dose-dependent toxicity in patients, and prolonged use may lead to the emergence of resistant pathogenic strains [17,26,27]. Colonisation of multi-drug-resistant bacteria transfers the antibiotic resistance genes to commensals or potential pathogens, thereby delaying the effect of antibiotics due to persistence of the resistance genes in the microbiota [28]. Topical or oral application of probiotics, such as Lactobacillus, could improve skin health by reducing skin colonisation or in ammation [29][30][31]; therefore, it is necessary to develop novel strains of probiotics with antimicrobial properties that are safe and sustainable.
L. plantarum strains APsulloc 331261 and 331266 isolated from green tea (Camellia sinensis) have been sequenced and annotated using whole-genome sequence analysis, and their safety and bene ts have also been described [32]. L. plantarum APsulloc 331261 alleviated gastric in ammation in an alcoholinduced gastric ulcer murine model [33]. Moreover, it markedly increased the abundance of various intestinal bene cial bacteria such as Bi dobacterium spp. and Clostridium butyricum [33]. Furthermore, L. plantarum APsulloc 331261-derived extracellular vesicles exerted anti-in ammatory effects on human skin by inducing macrophage polarisation [34].
In the present study, we examined the antibacterial and antifungal activity of L. plantarum APsulloc 331261 and 331266 against four strains (M. globosa, M. restricta, C. albicans, and C. acnes). Bacteria or fungi were incubated with L. plantarum APsulloc 331261 and 331266 using a plate agar overlay assay. We further validated the growth inhibition effects of L. plantarum APsulloc 331261 and 331266 against bacteria and fungi using a co-culture transwell assay.

L. plantarum APsulloc 331261 and 331266 inhibit the growth of Cutibacterium acnes and Candida albicans
The results of the agar overlay assay used to determine bacterial and fungal growth inhibition are summarised in Tables 2 and 3. L. plantarum strains APsulloc 331261 and 331266 signi cantly inhibited the growth of C. acnes or C. albicans. After 24 h of incubation, L. plantarum-C. acnes and C. albicans overlays revealed markedly strong zones of bacterial and fungal inhibition, respectively, around the L. plantarum APsulloc 331261 and 331266 colonies (Fig. 1). L. plantarum APsulloc 331261 inhibited the growth of C. acnes and C. albicans with ZDIs of 22.0 ± 1.7 and 23.0 ± 1.0 mm, respectively. In addition, C. acnes and C. albicans exhibited high sensitivity to L. plantarum APsulloc 331266 (ZDIs of 20.0 ± 2.0 and 27.0 ± 3.6 mm, respectively; Table 2). Furthermore, the 'R' value revealed the antibacterial and antifungal activity of L. plantarum APsulloc 331261 and 331266. The action of L. plantarum APsulloc 331261 presented 'R' values of 5.5 ± 1.3 and 23.0 ± 1.0 mm, whereas L. plantarum APsulloc 331266 had 'R' values of 4.3 ± 1.8 and 5.5 ± 1.7 mm against C. acnes and C. albicans, respectively (Table 3). No signi cant differences in growth inhibition were found between L. plantarum APsulloc 331261 and 331266.
For a more detailed evaluation regarding the growth inhibition of C. acnes and C. albicans in a co-culture system with L. plantarum, we conducted growth assays using a transwell chamber with a 0.4-µm porous membrane. These experiments revealed that C. acnes and C. albicans growth decreased in the presence of L. plantarum APsulloc 331261 and 331266 ( Fig. 2A and B). APsulloc 331261 reduced the growth of C. acnes and C. albicans by 61.2% and 77.7%, respectively, compared to the control. APsulloc 331266 decreased the growth of C. acnes and C. albicans by 70.2% and 70.1%, respectively, compared to the control. Despite the physical separation of the microorganisms, bacterial and fungal growth retardation may be caused by nutrient competition and diffusible inhibitors (e.g., lactic acid and bacteriocin) produced by L. plantarum APsulloc 331261 and 331266. Moreover, the growth inhibition assays using conditioned media (CM) derived from L. plantarum APsulloc 331261 and 331266 revealed that the growth of C. acnes and C. albicans was suppressed by L. plantarum culture supernatants in a dosedependent manner ( Fig. 2C and D). L. plantarum APsulloc 331261 and 331266 completely inhibited the growth of C. acnes in 100%-CM, while the two strains caused 35.4% and 40.0% growth inhibition in 50%-CM, respectively, compared to the control. Furthermore, L. plantarum APsulloc 331261 and 331266 signi cantly inhibited the growth of C. albicans by 68.1% and 75.8% in 100%-CM and 37.3% and 61.9% in 50%-CM, respectively, compared to the control.

L. plantarum APsulloc 331261 and 331266 inhibit the growth of Malassezia globosa and Malassezia restricta
The growth of dandruff-producing fungi M. globosa and M. restricta was observed in the absence or presence of L. plantarum APsulloc 331261 and 331266. Interestingly, the marked antifungal activity of L. plantarum APsulloc 331261 and 331266 was detected in a transwell co-cultured with fungus. A signi cant decrease in the growth of M. globosa and M. restricta was observed ( Fig. 3A and B). APsulloc 331261 reduced the growth of M. globosa and M. restricta by 71.1% and 72.3%, respectively, compared to the control. APsulloc 331266 decreased the growth of M. globosa and M. restricta by 77.6% and 75.9%, respectively, compared to the control. Moreover, CM derived from L. plantarum APsulloc 331261 and 331266 showed antifungal activity against M. globosa and M. restricta in a dose-dependent manner ( Fig. 3C and D). L. plantarum APsulloc 331261 and 331266 signi cantly inhibited the growth of M. globosa by 90.9% and 93.7% in 100%-CM and 80.5% and 87.5% in 50%-CM, respectively, compared to the control. Moreover, L. plantarum APsulloc 331261 and 331266 signi cantly inhibited the growth of M. restricta by 96.9% and 94.9% in 100%-CM and 89.3% and 89.7% in 50%-CM, respectively, compared to the control.
In general, these viability assay results provide strong evidence that L. plantarum strains APsulloc 331261 and 331266 can exert antibacterial and antifungal effects.

Discussion
Probiotics have recently become a popular microbiome therapy, and researchers are interested in exploring the mechanisms through which probiotic microorganisms interact with the body [35]. In addition to the commercially available probiotic strains, LAB from various natural sources are being isolated and screened to discover better probiotic strains [36]. In the present study, we examined the antibacterial (C. acnes) and antifungal (C. albicans, M. globosa, and M. restricta) activity of L. plantarum strains APsulloc 331261 and 331266 isolated from green tea, which have been previously described [32].
Probiotics and prebiotics have been widely evaluated for the treatment and prevention of infectious diseases due to their presence in the microbiome of several parts of the human body. In particular, studies in the eld of skin diseases, such as acne vulgaris, tinea pedis, dandruff, and candidiasis, have revealed the effect of probiotics following topical application or ingestion [45]. Probiotics or prebiotic cosmetics have been recently developed, which rebalance the composition of skin micro ora by inhibiting the growth of harmful pathogens while promoting the growth of bene cial bacteria among the resident microorganisms [46]. Kang et al. demonstrated the therapeutic effect of a lotion prepared as a concentrated powder (CBT SL-5) from Enterococcus faecalis SL-5-conditioned media on C. acnesinfected patients [47]. Muizzuddin et al. reported that L. plantarum extract (1% and 5%) reduced the size of acne lesions in clinical studies [30]. Jung et al. demonstrated that probiotic capsules (L. acidophilus, L. bulgaricus, and Bi dobacterium bi dum) signi cantly improved acne vulgaris by exerting a synergistic anti-in ammatory effect with systemic antibiotics and reduced the potential side effects of chronic antibiotic use [48]. Recently, L. plantarum IS-10506 isolated from dadih reduced clinical symptoms in children with mild and moderate atopic dermatitis [49].  [51].
Although the results from in vitro studies should be drawn carefully, our ndings further support the hypothesis that probiotic L. plantarum strains APsulloc 331261 and 331266 may ameliorate homoeostasis and microbial pro le in the skin.

Conclusion
The present in vitro study revealed that L. plantarum strains APsulloc 331261 and 331266 isolated from green tea could inhibit the growth of bacteria (C. acnes) and fungi (C. albicans, M. globosa, and M. restricta). The antibacterial and antifungal activity of L. plantarum APsulloc 331261 and 331266 was tested using two different in vitro methods. Future studies are warranted to investigate the effectiveness of L. plantarum APsulloc 331261 and 331266 as alternative therapeutics for eliminating antibiotic resistance and treating infection.

Fungal and Bacterial Strains and Culture Conditions
L.plantarum APsulloc 331261 and 331266 isolated from green tea (Osulloc farm, Jeju island, South Korea) were provided by AMOREPACIFIC (Yongin, Korea, KCCM11179P and KCCM11180P), and their safety and probiotic properties have been previously described [32]. L. plantarum APsulloc 331261 and 331266 were cultured at 37 °C for 24 h in MRS broth (Becton, Dickinson and Company; Sparks, MD, USA).
The fungal and bacterial strains used in this study were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and are listed in Table 1. C. albicans was cultured in yeast malt broth (Becton, Dickinson and Company) at 37 °C, on a 200 rpm shaking incubator for 24 h. M. globosa and M. restricta were cultured in modi ed Leeming & Notman agar (MLNA; ATCC medium 2737) without agar at 30 °C, on a 200 rpm shaking incubator for 24 h. C. acnes was cultured in modi ed reinforced Clostridium medium (ATCC medium 2107) under anaerobic conditions at 37 °C for 72 h. All fungal and bacterial strains were adjusted to an OD 600nm of 0.5 before use.
Growth inhibition using plate agar overlay assay The effects of the presence of L. plantarum APsulloc 331261 and 331266 on the growth of C. acnes and C. albicans were assessed on agar plates as previously described [52]. For the plate agar overlay assays, L. plantarum APsulloc 331261 and 331266 (4 μL of OD 600nm 0.5) were spotted on 1.5% MRS agar plates.
After incubation for 2 days at 37 °C, the plates were overlaid with C. acnes and C. albicans suspension (OD 600nm 0.5) in 0.7% MRS soft agar that was prepared after cooling to 40 °C in an autoclaved agar medium. Following solidi cation, the plates were incubated at 37 °C for 1 day in aerobic (C. albicans) or anaerobic (C. acnes) conditions. The zone diameter of inhibition (ZDI) value was determined, following the method reported by Shokryazdan et al. [53]

Statistical analysis
Data are presented as the mean ± standard deviation (SD). The control and test groups were compared using a paired t-test. Multiple comparisons were evaluated using one-way analysis of variance, followed by post-hoc t-tests. P-values < 0.05 were considered statistically signi cant.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.   added to the inserts of transwells, following which Cu. acnes (C) and C. albicans (D) were inoculated. After incubation at 37 ° for 24 h, the cell densities were determined at OD600nm. All data represent the mean ± SD. W** P < 0.01 vs. MRS.