Major diseases induced by pathogenic bacteria are dental caries and periodontitis, and the most common fungal disease in oral cavity is candidiasis.^1,2 According to epidemiological studies, dental caries is related with Streptococcus mutans and S. sobrinus, and periodontitis is caused by obligated anaerobic Gram-negative bacteria such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola,³ and oral candidiasis is related with Candida albicans.² Although various research have been studied on the characteristics of these pathogens and antibiotics and physical therapy to treat oral diseases, the studies on prevention of oral disease are still lacking. Recently, Oral biofilms are considered to be more important than individual characteristics of these bacteria in oral diseases, and comparative studies on characteristics between healthy biofilm and pathogenic biofilm is in progress.^4-6 Oral biofilms are classified according to the distribution of bacteria in the biofilm, and when the distribution of pathogenic bacteria is high levels, it is recognized as a disease-related biofilm, and when the distribution of normal flora is high levels, it is considered as a healthy biofilm.¹ Therefore, the ideal prevention of oral disease is to maintain a high distribution of commensal bacteria such as viridans streptococci except mutans streptococci within the biofilm.

Probiotics are live microorganisms and provide a health benefit to the host.⁷ Due to concerns about the emergence of antibiotics-resistant bacteria, many studies have been conducted on treatment and prevention of infectious disease using probiotics.⁸ Latobacilli plays an important role to maintain human healthy by stimulating native immune system and protection against infection of pathogenic bacteria.⁹ However, Since Lactobacillus species have characteristics of abundant production of lactic acid and aciduricity, it is careful when applying it to oral disease. Among probiotics, Streptococcus salivarius K12 is Gram-positive and facultative anaerobic bacteria. This bacterium, which is isolated from healthy person, is a pioneer colonizer of oral cavity.¹⁰ S. salivarius K12 is also known to be a probiotic bacterium and produces bacteriocin of two class such as salivaricin A2 and salivaricin B.¹¹ Above all, S. salivarius K12 don’t have aciduricity like Lactobacillus species. The selection of prebiotics as well as probiotics is important for treatment and prevention of disease. Prebiotics are defined “dietary prebiotics” as “a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health”.¹² Eventually, prebiotics for oral cavity can selectively influence oral microbiota, and it is necessary to find suitable prebiotics to enhance the growth and antimicrobial activity of S. salivarius K12

Although, S. salivarius K12 showed antimicrobial activity against oral bacteria,¹³ There are insufficient information on the culture conditions optimized to apply S. salivarius K12 to the oral cavity. The purpose of this study was to investigate effects of culture conditions on the growth and antibacterial activity of S. salivarius K12

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S. salivarius K12 was donated from Green store Inc. (Seongnam, Korea) and used in this study after isolating from Bactoblis^® tablet. S. salivarius K12 was cultivated with M17 broth (BD bioscience, Sparks, USA). For susceptibility assay, a species of Gram-positive and Gram-negative bacteria were selected, respectively, and Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277, and Candida albicans ATCC 10231 was selected for dental caries, periodontitis, and candidiasis, respectively. S. mutans was cultivated with Tryptic soy broth (BD bioscience, Spark, USA), and P. gingivalis was cultured with brain heart infusion (BHI; BD bioscience) supplemented with hemin (0.5 μg/ml; Sigmaaldrich Co., San Jose, USA) and vitamin K (0.2 μg/ml; Sigmaaldrich Co.). C. albicans was cultivated in tryptic soy broth (TSB; BD bioscience), The bacteria and fungus were maintained at 37°C in an aerobic condition (H₂ 5%, CO₂ 10%, and N₂ 85%) and an aerobic condition, respectively. In investigate characteristics of S. salivarius K12 by culture conditions, S. salivarius K12 was cultured with media of two types (medium containing animal or plant protein), and the components of two media is shown in Table 1. In another experiment, the medium containing plant protein was added with 10 mM sodium phosphate dibasic and 2 mM potassium phosphate, monobasic and then adjusted the level of pH 7.0 and pH 5.5 with lactic acid. S. salivarius K12 was cultured in the two conditions. The growth of S. salivarius K12 cultured in various conditions was measured optical density at 660 nm of wavelength every 2 hours. After collecting spent culture medium (SCM) of S. salivarius K12 cultured in various conditions, the SCM from various condition was investigated antimicrobial activity against oral bacteria according to Clinical and Laboratory Standard Institute (CLSI) guidelines. Briefly, 180 μl of the specific medium for each oral microbe was dispensed into 96-well plate (SPL Lifescience, Pocheon, Korea), and the SCM was added into the first row. The SCM was performed 2-fold serial dilution with micropipette. The bacterial suspensions were adjusted 2 × 10⁶ cells/ml for S. mutans, 3 × 10⁶ cells/ml for P. gingivalis, and 1 × 10⁶ cells/ml of Candida albicans after counting the bacteria using a bacterial counting chamber (Hausser Scientific, Horsham, USA). 20 μl of the bacterial suspension was inoculated into the prepared well with diluted SCM, and the plate was incubated at 37°C in an aerobic condition (H₂ 5%, CO₂ 10%, and N₂ 85%). The growth of bacteria was measured optical density at 660 nm of wavelength by a spectrophotometer (BioTek, Winooski, USA). The significant difference among each group was analyzed by non-parametrical analysis such as Kruskal Wallis test and Mann-Whitney U test using IBM SPSS statistics Ver. 23 (IBM, Armonk, USA). P-values less than 0.05 were considered statistically significant.

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When the growth of S. salivarius K12 using medium of animal protein and plant protein was investigated, although the growth of S. salivarius did not show significant difference between medium containing animal and plant protein, the its growth was slightly faster in presence of plant protein than animal protein (Fig. 1). The growth of S. salivarius K12 in Also, when the effects of pH level on the growth of S. salivarius K12 were examined, S. salivarius K12 was cultured very slow and did not reach stationary phase in the medium of pH 5.5 (Fig. 2). On other hand, the growth of S. salivarius K12 in pH 7.0 condition showed S shaped growth curve (Fig. 2). In case of S. salivarius K12 growth in two pH levels, there were a lot of growth differences to compare the antimicrobial activity of S. salivarius K12. Therefore, the antimicrobial activity of S. salivarius K12 cultured in medium containing animal or plant proteins was compared using SCM. The SCM of S. salivarius K12 cultured in medium containing animal and plant proteins significantly inhibited the growth of S. mutans at or above 4-fold diluted and 16-fold diluted concentration, respectively (P < 0.05) (Fig. 3A). The difference in antimicrobial activity of S. salivarius K12 cultured in the two media was approximately 4 times. Also, in case of examination of P. gingivalis, the SCM of the probiotics cultured in medium containing animal and plant protein significantly reduced the growth of P. gingivalis at or above 8-fold and 32-fold diluted concentration, respectively (P < 0.05) (Fig. 3B). Similar to the case of S. mutans , the two SCM of S. salivarius K12 showed 4 times difference in the antimicrobial activity against P. gingivalis. The SCM of S. salivarius K12 cultured in medium containing plant proteins significantly inhibited the growth of C. albicans at or above 4-fold diluted concentration (P < 0.05) (Fig. 4). The SCM from medium containing animal protein slightly inhibited the growth of C. albicans.

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S. salivarius K12 is a probiotic bacterium and isolated from the saliva of healthy child.¹⁴ This probiotic produces salivaricin A2 and salivaricin B that effectively inhibits the growth of S. pyogenes as a pharyngitis related bacterium.^15,16 Furthermore, S. salivarius K12 has the antimicrobial activity against oral bacteria and fungus and can colonize in oral cavity.^13,17 Due to these characteristics, S. salivarius K12 is considered to be more suitable for prevention and treatment of bacteria-related oral disease than other probiotics. However, there is still not enough information about the habit of S. salivarius K12. Therefore, in this study, the growth and antimicrobial activity of S. salivarius K12 according to the culture condition were investigated.

S. salivarius K12 showed faster growth in medium containing plant protein than animal proteins. Probiotics require nutrients and environments for colonization and growth like pathogenic bacteria because of protozoa. So, substances that help the function and growth of probiotics are called prebiotics, and oligosaccharide is representative. In this study, although it has not been examined exactly what component, it was observed that a protein derived from soybean, a representative plant protein, helps the growth of S. salivarius K12. This indicates that soy protein may be a prebiotic for S. salivarius K12. In experiment of pH levels, S. salivarius K12 was observed to grow very slowly in pH 5.5 as an acidic condition. Most bacteria produce lactic acid through carbohydrate metabolism and create the acidic environment. Even in a pH 5.5 environment, if S. salivarius K12 is active in carbohydrate metabolism, it can induce dental caries, but it is considered that S. salivarius K12 may slowly metabolize because the growth is slow in this environment. Therefore, this information supports that S. salivarius K12 is safe to apply for the prevention of oral diseases, and that substances with a pH buffer function may be included to increase the preventive effect of S. salivarius K12.

In order to investigate whether the two media affects only the growth of S. salivarius K12 or affects the antimicrobial and the antifungal activity of S. salivarius k12, the susceptibility of S. mutans and P. gingivalis was evaluated using the SCM of S. salivarius K12 cultured in medium containing animal or plant protein. S. mutans is a Gram-positive microbe and closely related with dental caries,⁷ and P. gingivalis is a Gram-negative anaerobe and a periodontopathogen.³ Both bacteria showed more susceptibility for the SCM from medium containing plant protein than animal protein. Furthermore, C. albicans appeared more susceptibility for the SCM from medium containing plant protein compared to animal protein

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The present study shown that the necessity of the condition to maximize the function of S. salivarius K12 as a probiotic. Eventually, in order to apply S. salivarius K12 to bacterial oral disease, co-substances may be needed for S. salivarius K12 to colonize in the oral cavity and exhibit stronger the antimicrobial activity.

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