|Year : 2020 | Volume
| Issue : 1 | Page : 33-40
Soy fermentation by orally isolated putative probiotic Streptococcus salivarius for healthy oral
Siew Ming Choo1, Ke Yee Yap1, Wei Hsum Yap1, Joo Ann Ewe2
1 Faculty of Health and Medical Sciences, School of Biosciences, Taylor’s University, Selangor, Malaysia
2 Healthcare Sector, Nano and Advanced Materials Institute Limited, Shatin, Hong Kong
|Date of Submission||31-Jul-2019|
|Date of Decision||11-Aug-2019|
|Date of Acceptance||09-Sep-2019|
|Date of Web Publication||25-Feb-2020|
Dr. Joo Ann Ewe
Nano and Advanced Materials Institute Limited, Units 517, 5/F, Lakeside 1, No. 8 Science Park West Avenue, Hong Kong Science Park, N.T
Dr. Wei Hsum Yap
No. 1 Jalan Taylors, Subang Jaya 47650, Selangor Darul Ehsan
Source of Support: None, Conflict of Interest: None
Aim: Consumption of aglycones-rich fermented soy has been positively associated with the recuperation of systemic inflammation. As oral conditions have an impact on overall health and diseases, the reestablishment of bacteria particularly by indigenous oral probiotic is believed to be able to reverse oral inflammation by antagonize oral pathogen and thus maintaining good oral health. Streptococcus salivarius can be combined with bioactive isoflavone aglycones to further improve oral healthiness. This study aimed to explore decent advantages by indigenous source of Streptococcus isolated from healthy periodontal to ferment soy while maintaining probiotic properties before delivering in product targeting oral health. Materials and Methods: Putative probiotic properties of isolated strains were explored through antibiotic susceptibility ensuring safe strain consumption. S. salivarius from Taylor’s University Culture Collection (TUCC) 1254 was selected for probiotic properties evaluation, in comparison with the type strain. Both strains were evaluated for fermentation ability in soy through growth, β-glucosidase assay, and isoflavone bioconversion. Results: The antagonistic ability of S. salivarius TUCC 1254 was better under anaerobic than microaerobic condition; the strain also showed higher colonizing ability to epithelial tissue than the tooth surface resemblance. The strain showed vast cell growth accompanied by organic acid production and higher β-glucosidase enzyme to bioconvert isoflavone glucosides to bioactive aglycones that could reduce oral inflammation than K12. Soy improved auto-aggregation ability of S. salivarius, which could promote biofilm formation and thus could achieve enhanced oral pathogen-eradicating intention. Conclusion: This study suggested that indigenous bacteria could serve as a better source of oral probiotics and coupling with fermented soy bioactive isoflavone aglycones, become a deliverable platform for improving oral health.
Keywords: Aglycones, Oral Health, Periodontopathogens, Soy Fermentation, Streptococcus Salivarius
|How to cite this article:|
Choo SM, Yap KY, Yap WH, Ewe JA. Soy fermentation by orally isolated putative probiotic Streptococcus salivarius for healthy oral. J Int Oral Health 2020;12:33-40
|How to cite this URL:|
Choo SM, Yap KY, Yap WH, Ewe JA. Soy fermentation by orally isolated putative probiotic Streptococcus salivarius for healthy oral. J Int Oral Health [serial online] 2020 [cited 2020 Apr 3];12:33-40. Available from: http://www.jioh.org/text.asp?2020/12/1/33/279219
| Introduction|| |
Soy contains high nutritional value needed to support the growth of most probiotics, particularly, lactic acid bacteria (LAB) of genus Lactobacillus and Bifidobacterium. Despite the major sources of probiotics are of gut origin, the concept of probiotics applies to oral health maintenance, and it is believed that an indigenous source could ensure decent advantage. Streptococcus salivarius is one of the most thoroughly studied oral probiotic species due to its presence throughout our lives in the oral cavity. Among the strains of S. salivarius, K12 is the first commercialized oral probiotic and has also been reported to reduce symptoms of halitosis and oral thrush. Lactobacilli isolated from the oral cavity of healthy people have been reported to possess probiotic potential as a result of their adaptive and probiotic properties. Thus, it is worth exploring the diverse pool of beneficial bacteria from healthy host periodontal to expand oral health application. To date, no study exploring healthy periodontal isolated Streptococcus as putative probiotics in antagonizing oral pathogens is available. LABs probiotics produce β-glucosidase enzyme, which can hydrolyze the non-bioactive β-glucosidase into biologically active aglycones during soy milk fermentation. Daidzin, genistin, and glycitin are the three main isoflavone glucosides, which will be converted into their respective bioactive aglycones, namely daidzein, genistein, and glycitein. Fermented soy milk has been found to show bactericidal activity both in in vitro and animal models, attributable to the enhanced aglycones that inhibit bacterial growth. Oral infections always begin with the formation of biofilms where communities of opportunistic pathogens are able to colonize. Considering the joint benefits of oral probiotics and the increased level of aglycones on soy fermentation, the putative probiotic with known efficacy could enhance the effectiveness of antimicrobial therapies for the management of oral health.
| Materials and Methods|| |
Subject recruitment and saliva collection: A total of 22 healthy subjects (aged 18–35 years) that fit the inclusion criteria, which included healthy periodontal, not on antibiotics, probiotic products, and chlorhexidine mouthwash during and two weeks before the intervention were included in this study. The protocol of this study was approved by the University Human Ethics Committee (HEC 2017/037). Informed written consent was acquired from each subject that agreed to participate voluntarily. Potential strains of Streptococcus were isolated from the saliva of subjects with healthy periodontal condition. Saliva was collected using Super SAL according to the manufacturing instruction (Universal Saliva Collection Device, Oasis Diagnostics, Vancouver, USA). S. salivarius K12 that was isolated from tablets (BLIS Technologies, Dunedin, New Zealand) was used as a control for subsequent analyses.
Microbial isolation and identification: Decimal dilutions of saliva samples were cultured on Mitis Salivarius (MS) selective agar (HiMedia, Mumbai, India) supplemented with 1% potassium tellurite (HiMedia) to isolate strains of Streptococcus. Fifteen irregular-shaped light blue colonies with hardened texture on selective agar were picked for gram staining and catalase test as preliminary confirmation. Following this, the identification of isolated Streptococcus was performed according to the Biolog’s Protocol carried out with some minor modification. Results were read and interpreted by the identification system’s software (GEN III OmniLog Database, version 5.2.1, Hayward, California).
Antibiotic susceptibility test assay: Antibiotic susceptibility test was performed according to the study conducted correlated with the Clinical and Laboratory Standards Institute 2012 (https://clsi.org/) guidelines with some modification. Antibiotic discs containing respective effective concentrations of penicillin, gentamicin, tetracycline, erythromycin, and clindamycin were placed on the bacteria inoculated plate and incubated at 37°C for 24h. It was tested along with negative control strains Staphylococcus aureus American Type Culture Collection (ATCC) 29737 and Escherichia More Details coli ATCC 25922. The diameters of the zone of inhibition were measured, and the results were interpreted.
Antagonistic effect of S. salivarius against oral pathogens: The antagonistic activities of S. salivarius against halitosis causing bacteria, namely Fusobacterium nucleatum ATCC 10953, clinically isolated Treponema denticola and Streptococcus mutans were investigated. One hundred µL of the activated pathogens with the optical density of 0.3 (at 600nm) were uniformly spread on Tryptic Soy Agar (Merck, New Jersey, United States) supplemented with defibrinated sheep blood. Agar wells of diameter 5mm were cut using sterile cork borer and 20 µL of activated S. salivarius suspension were placed into each agar well and incubated under microaerobic (CO2 incubator, Memmert, Schwabach, Germany) and anaerobic conditions (anaerobic chambers, Whitley DG250 Anaerobic Workstation, West Yorkshire, UK) at 37°C for 24h. Growth inhibitory activity of S. salivarius was recorded by measuring the clear zone around, namely inhibition zone diameter.
Adherence of S. salivarius to human oral squamous cell and hydroxyapatite (HA): The adherence ability of S. salivarius was evaluated on both human oral squamous cell carcinoma (HSC3) cells and HA. The assay for bacterial adhesion to HSC3 cells was performed. Adherence of isolated bacteria toward HA was carried out as described.
Fermentation of soy medium: To assess the suitability of soy as a carrier, its potential was evaluated through pH changes. Soy medium (4% wt/vol) was prepared using soy powder (VIS Foodtech Ingredient Supplies, Kuala Lumpur, Malaysia). Five percent (vol/vol) activated S. salivarius were aseptically inoculated into soy medium, and the samples were drawn at intervals of 0, 6, 12, 18, and 24h of fermentation at 37°C for pH determination using calibrated pH-meter (Eutech Instruments, Singapore). Cell viability was performed at interval of 0, 12, and 24h.
Determination of β-glucosidase activity: The intracellular β-glucosidase activity of S. salivarius was determined. It was measured by the rate of hydrolysis of 4-nitrophenyl β-d-glucopyranoside as a substrate (pNPG; Sigma-Aldrich, St. Louis, Missouri). The protein concentration of the crude enzyme extract was determined using bovine serum albumin as standard. The specific activity was expressed as milliunits (mU) of β-glucosidase activity per milligram of protein.
Determination of isoflavones in soy milk: The concentration of isoflavones was determined using a reversed-phase high performance liquid chromatography (HPLC) method for unfermented and fermented soy milk after extraction. The HPLC-grade isoflavone standards including daidzin, daidzein, glycitin, glycitein, genistin, and genistein (Sigma-Aldrich, Missouri, United States). The concentration of isoflavones was identified using Shimadzu HPLC (Shimadzu, Japan) fitted with an Inertsil ODS-3 column (150 × 3mm, 5mm, GL Sciences, Tokyo, Japan). The mobile phase consisted of solvent A (water:phosphoric acid, 1000:1, vol/vol) and solvent B (water:acetonitrile:phosphoric acid, 200:800:1, vol/vol/vol) with a flow rate of 1 mL/min in a concentration gradient. The gradient elution was started with solvent A in the sequence of 95% (2min) → 65% (29min) → 50% (31min) → 95% (40min) → 95% (43min). The separation was operated at 40°C, and the detection wavelength was at 259nm.
Aggregation ability of Streptococcus on soy medium fermentation: Briefly, S. salivarius was harvested from soy medium after 24h of fermentation at 37°C. Bacteria suspension in soy medium was then transferred to soy agar (4% wt/vol) plates and incubated at 37°C for 24h. Bacteria colonies were then resuspended in 0.15mol/L sodium chloride solution and incubated at 37°C for 24h. Samples were carefully drawn out without shaking at 0, 6, and 24h to measure the optical density using UV/VIS spectrophotometer (Thermo Scientific, Massachusetts, United States) at 600nm. The aggregation percentage (AP) was measured by using the following formula:
where, ODt (t = 6 or 24h) and OD0 (t = 0h) indicate the optical density of final and initial times, respectively.
Statistical analyses: The Statistical Package for the Social Science Program (SPSS, Chicago, USA), version 20, was used to analyze the data obtained from the tests carried out. Data were compared using one-way analysis of variance, unless otherwise stated. Mean value comparisons were performed using Tukey test. Data presented were mean ± standard deviations from two separate runs with a significance level of α = 0.05.
| Results|| |
The 15 strains of Streptococcus isolated in selective medium from healthy subjects were gram positive and catalase negative, with six species of Streptococcus (as in [Table 1]) and one Actinomyces neuii on Biolog identification. Actinomyces neuii and Streptococcus hyointestinalis were eliminated from analyses as they were not orally found commensal bacteria. This indicated the distinctiveness of bacterial flora in oral cavity of every subject.
Strains of Streptococcus showed different profiles of antibiotic resistance [Table 1]. The isolated strains, S. salivarius TUCC 1254, were susceptible to penicillin, tetracycline, erythromycin, and clindamycin, and an intermediate susceptibility toward gentamicin. To ensure safe consumption, we chose this strain for subsequent probiotic qualities evaluation.
In general, the streptococci showed antagonistic activity against the common resident harmful pathogens that can lead to oral disorders such as periodontitis and halitosis,, the F. nucleatum ATCC 10953, clinically isolated T. denticola and S. mutans [Table 2]. Antagonistic activity of S. salivarius against oral pathogen was observed under both microaerobic and anaerobic environment to resemble the ecological niche environment such as tooth surface and gingival epithelium in oral cavity. Particularly, antagonism ability of the isolated strain S. salivarius TUCC 1254 was better in the absence of oxygen in which the inhibitory activity of this strain toward F. nucleatum, T. denticola, and S. mutans was 18.2% (P = 0.017), 72.7% (P = 0.000), and 69.7% (P = 0.000), respectively, higher under anaerobic than microaerobic condition.
|Table 2: Zone of inhibition (mm) of Streptococcus salivarius against periodontal pathogens by agar-well diffusion method|
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Strains of S. salivarius showed varying levels of adherence toward the HSC3 cell line and HA surface [Table 3]. The strains showed better adhesion to HSC3 cell line (34.5%–83.85% higher, P = 0.000) than HA. S. salivarius TUCC 1254 showed higher adherence potential to oral epithelial tissue such as the tongue and gingival epithelial rather than that to teeth, which thus stands a better chance in eliminating oral pathogens via its antimicrobial ability as shown through antagonistic study [Table 2].
|Table 3: Adhesion of Streptococcus salivarius to human oral squamous cell carcinoma and hydroxyapatite|
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A significant drop of pH in soy media (P = 0.000) in the first 12h on fermentation by the streptococci. The results indicated both S. salivarius K12 and TUCC 1254 actively multiplying while producing higher amount of organic acids during the first 12h of exponential growing before entering stationary phase.
Both strains of S. salivarius were able to grow well in soy medium [Figure 1]. S. salivarius TUCC 1254 increased in twofold on fermentation and showed better growth than S. salivarius K12 in soy medium (P = 0.000). This corresponded well with the reduction of pH in growth medium after 24h (a 9.6% dropped for S. salivarius TUCC 1254; a 7.8% dropped for S. salivarius K12). Such observation reflects niche adaptation capability of bacteria from healthy oral to soy by producing different key enzymes in response to dietary change.
|Figure 1: Growth of Streptococcus salivarius K12 (squares) and S. salivarius Taylor’s University Culture Collection (TUCC) 1254 (diamonds) in soy medium for 24h at 37°C. Error bars SEM (n = 2). *Mean values are significantly different (P < 0.05) via independent t-test|
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There was an increase in titratable acidity of soy media on fermentation by bacteria but the results were strain dependent [Table 4]. S. salivarius K12 and TUCC 1254 produced comparable amounts of organic acids where the pH of the fermented media dropped from 7.4 to 6.7−6.8 [Figure 2].
|Table 4: Titratable acidity of Streptococcus salivarius-fermented soy media upon fermentation for 24h at 37°C|
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|Figure 2: Changes of pH in Streptococcus salivarius fermented soy media over 24h at 37°C for S. salivarius K12 (squares) and S. salivarius Taylor’s University Culture Collection (TUCC) 1254 (diamonds). Error bars SEM (n = 2). Mean values between the two strains are statistically insignificant (P > 0.05) via independent t-test|
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S. salivarius TUCC 1254 had significantly higher (P = 0.097) β-glucosidase activity compared to S. salivarius K12 [Table 5], indicating higher efficiency of indigenous bacteria than that of commercially available for potential application.
|Table 5: β-Glucosidase activity from cell extracts of Streptococcus salivarius on fermentation in de Man, Rogosa, and Sharpe media at 37°C for 24 h|
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Fermented soy milk contained higher amount of isoflavone aglycones than glucosides in unfermented soy milk [Table 6]. The concentration of aglycones (daidzein, glycitein, and genistein) in soy milk fermented by S. salivarius TUCC 1254 was substantially (P = 0.000) higher than that by S. salivarius K12, attributable to the higher β-glucosidase activity.
|Table 6: Concentrations of isoflavones in unfermented soymilk and Streptococcus salivarius-fermented soy milk on fermentation at 37°C for 24 h|
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The adhesion of Streptococcus has enhanced on growing in soy as shown by the auto-aggregating ability of the cells with prolong incubation from 12th to 24th h [Figure 3]. Both strains aggregated better at 24th h than at 12th h (S. salivarius K12: P = 0.000; TUCC 1254: P = 0.000). S. salivarius K12 appeared to consistently having better aggregation than TUCC 1254 at the 12th h (P = 0.004) and the 24th h (P = 0.005). Such interaction of bacteria to promote aggregation with oral probiotic is important as this could inhibit or reduce the dental plaque formation.
|Figure 3: Aggregation percentage of Streptococcus salivarius K12 (squares) and S. salivarius Taylor’s University Culture Collection (TUCC) 1254 (diamonds) after 12 and 24h incubation at 37°C. Results are expressed as mean values ± standard deviation, Error bars, standard error of mean (SEM) (n = 2). *Indicated significantly different (P < 0.05) via paired t-test|
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| Discussion|| |
Microflora of the healthy cavity had been profiled a decade ago where it indicates that distinctive species usually colonizes with site and subject specific. With specific characteristic features of S. salivarius colony on the selective MS agar, which revealed irregular-shaped light blue colonies with hardened texture and gum drop appearance, they can be easily isolated.
There has been increasing interest in the use of probiotics for the management of oral infections. Burton et al. reported that only 60% of subjects in the study possessed S. salivarius strain in their saliva before probiotic intervention. Specific screenings for selecting appropriate strains are crucial to ensure the functionality and safety of the newly isolated strains. Despite studies about probiotic for oral management has been increasing tremendously, till date, guidelines outlining probiotic properties are of gastrointestinal health point of view. Regardless of target sites, the mechanism of actions recommended for oral probiotics resembles gastrointestinal species. For instance, strains safety, adhesion ability, and antagonism activity toward pathogenic strains.
Antibiotics have been used extensively in the treatment of bacterial infection, and the emergence of antibiotic-resistant strains has posed a serious threat for the future. The safety issue of the putative probiotic is of special concern as there is rapid expansion of probiotic market, which leads to increased probiotic consumption in the society., The antibiotic susceptibility profile of the chosen strain S. salivarius TUCC 1254 resembled S. salivarius K12 with a low possibility of harboring antibiotic-resistant genes, thereby it is recommended to be incorporated as food adjunct.
Antagonistic test was performed in the attempt of replacing bacteria responsible for oral disorders with isolated bacteria, to establish a balance ratio in the oral microbial community to achieve oral well-being. It is believed that the orally isolated S. salivarius could provide a potential pool of putative probiotic-targeting oral cavity. The distinct environmental conditions of anaerobic and microaerobic cultivation have been found to cause suppression of different pathogens by probiotic bacteria through acidification of cytoplasm in the pathogenic strains. The oxygen tension during incubation has affected the growth and secretion of organic acids and bacteriocin production by certain species. Results obtained from our tested strains showed higher inhibition zone when cultivated under anaerobic condition. We postulated that the secretion of organic acids and bacteriocins in S. salivarius studied against pathogens could have been induced more under anaerobic condition for better inhibition.
The ability to adhere to the host cell surfaces has been suggested to be a crucial selection criterion to be used as probiotics. The high adhesion capacity of the bacteria is presumably due to the surface adhesion where S. salivarius had been reported to possess multiple adhesive strategies that enable it to bind on various biological surfaces. The binding of S. salivarius to human cell is mediated by the specific interactions between the adhesins and the complementary receptors. Glycoprotein AgC and a 27kDa protein are the components that promoted the binding of S. salivarius to oral epithelial cells and salivary–glycoprotein EP–GP, respectively. The proficiency of bacterial adhesins has been found to be affected by the differences in the level of charge, hydrophobicity, and structure of cell surface of bacteria. Thus, S. salivarius TUCC1254 could have possessed the adhesin that enabled it to adhere and colonize better to oral epithelial tissue, which then antagonized the anaerobic halitosis-causing oral pathogens.
It seems feasible to incorporate S. salivarius isolated from the original habitat in human oral cavity as this strain is most prevalent in healthy subjects.Streptococcus is a facultative anaerobe. It could metabolize the carbon source in soy medium and cause reduction of pH level. As there is no history of using S. salivarius for soy fermentation, medium acidity was first used as an indicator to assess the growing pattern of Streptococcus in soy medium. Soluble sugars can be found in soy, including stachyose, raffinose, sucrose, glucose, galactose, and fructose. Type strain S. salivarius K12 has been reported to carry oxidase reaction enzyme, the β-glucosidase, and has been found to be able to hydrolyze d-raffinose. The marginal increase in organic acid [Table 4] and slight drop in pH [Figure 2], yet enormous growth of S. salivarius [Figure 1] in soy, which was observed in this study, could be attributable to its ability to produce key enzymes to use carbohydrate sources in the growth medium as a result of niche adaptation.
Till date, no study has been conducted to analyze the β-glucosidase activity on S. salivarius in fermented soy milk. However, the presence of enzyme β-glucosidase in S. salivarius K12 was confirmed in a submitted generally recognized as safe (GRAS) exemption claim report to food and drug administration. The indigenous S. salivarius TUCC 1254 produced higher amount of β-glucosidase that led to production of higher bioactive aglycones that was bioavailable on fermentation. Such observation indicates the prospect of using native source could offer better anticipation.
Auto-aggregation measurements are widely used as an indirect method for evaluating the adhesion capacity of bacteria, including potential probiotics. Oral infections always begin with the formation of biofilms where communities of microorganisms gather together with a slimy matrix. Observation from the results suggested longer incubation period, which resulted in higher AP. The increased aggregation of S. salivarius on prolonged incubation in soy has suggested that the changes of surface properties were due to growth media, which enabled the bacteria to form biofilm for colonization but prohibited the deposition of pathogens locally.
Taken together, this study successfully identified the orally isolated S. salivarius TUCC 1254 as a putative probiotic candidate with known efficiency, particularly anti-oral pathogens potential. This β-glucosidase enzymes possessing strain enhanced aglycones content is expected to improve oral health, including reducing the occurrence of halitosis. Although the presence of resistance genes among the strains of Streptococcus indicates the need of in-depth research on the characterization of the resistant determinants, which is crucial so as to reduce the risk of antibiotic resistance development. It is believed that the subsequent study on oral health improvement would benefit from using the S. salivarius TUCC 1254-fermented soy as the medium for in vivo trial.
Ethical policy and institutional review board statement
This study did not involve clinical trial. Ethical approval was obtained for the collection of saliva samples from healthy subjects voluntarily. This study was approved by Taylors’ University Human Ethics Committee (TUHEC) on December 19, 2017. All the procedures have been performed as per the ethical guidelines laid down by the Declaration of Helsinki (1964).
Data availability statement
The data set used in this study is available on request from Dr. Ewe Joo Ann (email@example.com).
We would like to thank Professor John Tagg for sponsoring Streptococcus salivarius K12 used in this study and Professor Song Keang Peng for advising about the oral pathogens antagonistic study.
Financial support and sponsorship
This work was supported by the Ministry of Higher Education, Malaysia via Fundamental Research Grant Scheme (FRGS) (FRGS/1/2014/SG05/TAYLOR/03/2) and Taylor’s University through its Postgraduate Research Scholarship Program.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]