Research Article
Volume 1 Issue 5 - 2015
Stevioside Hydrate Effect on Growth, Acidogenicity and Adhesion of Streptococcus Mutans In Vitro
Anat Baniel1*, Sarit Faibis1, Doron Steinberg2 Nili Tickotsky3 and Moti Moskovitz1
1Department of Pediatric Dentistry, Hebrew University, Israel
2Institute of Dental Sciences, Hebrew University, Israel
3Bar Ilan University, Israel
*Corresponding Author: Anat Baniel, Department of Pediatric Dentistry, Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel.
Received: May 12, 2015; Published: June 04, 2015
Citation: Anat Baniel., et al. “Stevioside Hydrate Effect on Growth, Acidogenicity and Adhesion of Streptococcus Mutans Invitro”. EC Dental Science 1.5 (2015): 188-192.
Background and aim: The present study examined the in vitro effect of pure glycoside Stevioside hydrate on pH, growth and biofilm formation of Streptococcus mutans.
Method: S. mutans were supplemented with different concentrations of Stevioside hydrate. Bacterial growth, metabolism and effect on biofilm formation were examined.
Results: The presence of Stevioside hydrate had no significant effect on: 1) S. mutans growth; 2) Total bacterial growth; 3) S. mutans' acidogenicity; 4) S. mutans adhesion in biofilm formation.
Conclusion: Stevioside hydrate has no cariogenic potential as demonstrated by acidogenicity, S. mutans growth and biofilm effect.
Keywords: Stevioside hydrate; Streptococcus mutans; Biofilm; acidogenicity
The effect of various sweeteners on oral health and caries formation is well known. Following the establishment of the negative effect of sucrose on oral health and caries formation, most studies have focused on the effect of non-cariogenic sugar substitutes such as Sorbitol and Xylitol [1]. Various studies conducted around the world (mostly in children) demonstrated a 30-60% decline in caries formation following the use of chewing gum and dentifrice containing these sugar substitutes [2-8]. Xylitol has been demonstrated to be an effective sugar substitute, and the use of Xylitol or Sorbitol (or a combination of the two) as sucrose substitutes was proven to substantially lower caries rate [1].
Stevia is a calorie-reduced sweetener derived from the South American plant Stevia rebaudiana. The Steviol glycosides Stevioside and Rebaudioside A are the major components of Stevia and are the main contributors to its extremely sweet taste. The two basic forms of the Stevia plant, Stevioside or Stevia extract, are commonly used as sugar substitutes and as components of dentifrices and mouth rinses in South America and the Far East (Korea, Thailand, China and Japan). Recently there has been increasing interest in the use of this plant extract as a sugar substitute, due to its low caloric content which may reduce common effects of sucrose consumption such as obesity and diabetes mellitus. Moreover, this natural herb has fewer potential side effects than synthetic sweeteners.
Stevia-based sweeteners are approved for consumption as food additives in Brazil, South Korea and Japan. However, Stevioside and its derivatives are not yet considered Generally Recognized as Safe (GRAS) by the FDA, and are therefore categorized as dietary supplements in the US as well as in the EU. In 2006 the Joint FAO/WHO Expert Committee on Food Additives (JECFA) temporarily approved its Acceptable Daily Intake (ADI), which is currently up to 5.0 mg per kg of body mass.
According to the JECFA tests in 1999 no carcinogenic effects were found for an intake of no more than 2g per kg of body mass in men and 2.4g per kg of body mass in women [9]. Most studies agree that a reasonable daily intake of Stevioside (5 mg per kg of body mass) is safe and is neither carcinogenic nor teratogenic [10-13].
Dental caries is a significant health problem in many countries. Cariogenic bacteria produce acid due to fermentation of simple diet carbohydrates as sucrose. Mutans streptococci (MS) is one of the leading pathogens responsible for human caries. Prevention of dental caries would greatly benefit from the eradication of MS from the oral flora, an impairment of its ability to adhere to dental surfaces, or a reduction of its acidogenicity. In accordance with these goals, the aim of the present study was to determine the effects of various concentrations of Stevioside hydrate extracts (the pure extract of Stevia) on the pH, growth and biofilm formation of Mutans streptococci in vitro.
Materials and Methods
All experiments were conducted on S. mutans 27351 ATCC grown overnight in brain heart infusion (BHI) broth (Difco Laboratories, Maryland, USA) at 37°C in air atmosphere supplemented with 5% CO2 (v/v). The bacteria were grown in BHI supplemented with different concentration of Stevioside hydrate, (Sigma, St. Louis, MO, USA) with or without 2% Sucrose. After an overnight incubation, rate of growth of bacteria was determined by light absorbance at 450 nm (Spectrophotometer, Camspec M302, Cambridge, UK).
Stevia's effect on bacterial metabolism was estimated through measurement of changes in media pH. Measurements were performed following incubation, using pH level indicator test strip (pH indicator strips, Mark, KGaA, Darmstadt, Germany).
Evaluation of the effect of Stevia on biofilm formation was performed using the crystal violet technique [14]. After incubation the growth media was removed and the biofilm was rinsed carefully with 1.0 ml of PBS. Biofilm on the tube walls was stained using 1% crystal violet. After 15 minutes the stained biofilm was rinsed thoroughly with PBS. The dye was then extracted using 70% ethanol, and the intensity of the dye absorbed to the biofilm was determined using a 595 nm Genius spectrophotometer (Genius, Tecan, Männedorf, Switzerland)
Data was analyzed using SPSS software version 20. The analysis consisted of Kruskal-Wallis non parametric tests. Significance level was set at p ≤ 0.05.
Kruskal-Wallis test showed no differences between S. mutans growth rate on the various Stevioside hydrate concentrations as compared to the control (P = 0.12) (Figure 1). Total bacterial growth with 2% sucrose addition to the media was also not affected by the presence of Stevioside hydrate (P = 0.08) (Figure 2). Stevioside hydrate had no significant effect on S. mutans adhesion during biofilm formation (P = 0.12) (Figure 3). S. mutans acidogenicity was not influenced by the presence of Stevioside hydrate at the tested concentrations, with the pH remaining identical throughout the different concentrations.
Figure 1: The effect of Stevioside hydrate on bacterial growth.
OD on the Y axis signifies Optical Density.

Figure 2: The effect of different concentrations of Stevioside hydrate and 2% sucrose on bacterial growth in BHI.
OD on the Y axis signifies Optical Density.

Figure 3: Biofilm crystal violet stain- Crystal violet stain of S. Mutans biofilm in the presence of different concentrations of Stevioside hydrate and 2% sucrose.
OD on the Y axis signifies Optical Density.
Stevia is a natural non sugar substitute used as calorie free ingredient. Few studies have explored its effect on oral bacteria and most of them had used Stevia extracts [15,16] rather than the pure compound of Stevioside hydrate. The present study's distinctiveness is in the use of pure Stevioside hydrate. The decision to use pure Stevioside hydrate was based on the concept that only the use of purified Stevioside hydrate, Steviol or Rebaudioside A may enable significant progress in the investigation of those ingredients' affect on the oral flora.
The present study demonstrated that Stevioside hydrate did not influence S. mutans' growth: it neither promoted S. mutans' growth nor inhibited it. Stevioside hydrate also did not influence the acidogenicity of those bacteria, as the pH values after bacterial incubation did not change with or without Stevioside hydrate. In addition, Stevioside hydrate did not induce nor inhibit biofilm formation.
The present outcomes support those obtained by Zanela., et al. [15], who found that Stevia and fluoride mouth rinses had no anti-biofilm effect, and also no effect on S. mutans and S. sobrinus salivary levels. Das., et al. [16] found that 0.5% Stevioside and 0.5% Rebaudioside A had no cariogenic effect and no effect on S. sobrinus counts in rats. Mouth rinses containing Rebaudioside A were not found more acidogenic than water or sucralose in humans [17]. The results of the present study are in accordance with those studies.
The results obtained in this in vitro study indicate that Stevioside hydrate, the active glycoside of Stevia extract, has no cariogenic potential as depicted by acidogenicity, S. mutans' growth and biofilm effect. Unlike Xylitol, Stevioside hydrate has no adverse cariogenic effect such as bacterial growth or acid generation due to fermentation. In addition, it has no positive or negative effect on biofilm formation. Further studies with pure Stevioside hydrate are necessary to determine the effect of this substance on other cariogenic bacteria.
Stevioside hydrate has no cariogenic potential as demonstrated by acidogenicity and no effect on S. mutans growth and biofilm formation.
Bullet Points
The sweetener Stevioside hydrate has no cariogenic potential as demonstrated by acidogenicity and no effect on S. mutans growth and biofilm formations.
  1. Hayes C. “The effect of non-cariogenic sweeteners on the prevention of dental caries: a review of the evidence”. Journal of Dental Education 65.10 (2001): 1106-1109.
  2. Beiswanger BB., et al. “The effect of chewing sugar-free gum after meals on clinical caries incidence”. The Journal of the American Dental Association 129.11 (1998) : 1623-1626.
  3. Mäkinen KK., et al. “Polyol-combinant saliva stimulants and oral health in Veterans Affairs patients-an exploratory study”. Special Care in Dentistry 16.3 (1996): 104-115.
  4. Mäkinen KK, et al. “Xylitol chewing gums and caries rates: a 40-month cohort study”. Journal of DentalResearch 74 (1995): 1904-1913.
  5. Mäkinen KK. , et al. “Polyol chewing gums and caries rates in primary dentition: a 24-month cohort study”. Caries Research 30.6 (1996): 408-417.
  6. Mäkinen KK., et al. “Stabilisation of rampant caries: polyol gums and arrest of dentine caries in two long-term cohort studies in young subjects”. International Dental Journal 45.1 suppl 1 (1995): 93-107.
  7. Alanen P., et al. “Xylitol candies in caries prevention: results of a field study in Estonian children”. Community Dentistry and Oral Epidemiology 28.3 (2000): 218-224.
  8. Petersen PE and Razanamihaja N. “Carbamide-containing polyol chewing gum and prevention of dental caries in schoolchildren in Madagascar”. International Dental Journal 49.4 (1999): 226-230.
  9. Joint FAO/WHO Expert Committee on Food Additives. “Toxicological Evaluation of Certain Food Additives. Stevioside”. WHO Food Additives Series 42 (1999): 119-143.
  10. Yamada A., et al. “Chronic toxicity of dietary Stevia Extracts”. Journal of the Food Hygienic Society of Japan 26 (1985): 169-183.
  11. Xili L., et al.“Chronic oral toxicity and carcinogenicity study of stevioside in rats”. Food and Chemical Toxicology 30.11 (1992): 957-965.
  12. Toyoda K., et al. “Assessment of the carcinogenicity of stevioside in F344 rats”. Food and Chemical Toxicology 35.6 (1997): 597-603.
  13. Hagiwara A., et al. “Effects of three sweeteners on rat urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)-nitrosamine”. Gan 75.9 (1984): 763-768.
  14. Mowat E., et al. “Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigates”. Journal of Medical Microbiology 56.Pt 9 (2007): 1205-1212.
  15. Zanela NL., et al. “The influence of mouth rinses with antimicrobial solutions on the inhibition of dental plaque and on the levels of mutans streptococci in children”. Pesquisa Odontologica Brasileira 16.2 (2002): 101-106.
  16. Das S., et al. “Evaluation of the cariogenic potential of the intense natural sweetners stevioside and rebaudioside A”. Caries Research 26.5 (1992): 363-366.
  17. Goodson JM., et al. “Effect of a Truvia® Rebiana on the pH of Dental Plaque”. Abstract presented at the International Association for Dental Research General Sessions, Barcelona (2010).
Copyright: © 2015 Anat Baniel., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PubMed Indexed Article

EC Pharmacology and Toxicology
LC-UV-MS and MS/MS Characterize Glutathione Reactivity with Different Isomers (2,2' and 2,4' vs. 4,4') of Methylene Diphenyl-Diisocyanate.

PMID: 31143884 [PubMed]

PMCID: PMC6536005

EC Pharmacology and Toxicology
Alzheimer's Pathogenesis, Metal-Mediated Redox Stress, and Potential Nanotheranostics.

PMID: 31565701 [PubMed]

PMCID: PMC6764777

EC Neurology
Differences in Rate of Cognitive Decline and Caregiver Burden between Alzheimer's Disease and Vascular Dementia: a Retrospective Study.

PMID: 27747317 [PubMed]

PMCID: PMC5065347

EC Pharmacology and Toxicology
Will Blockchain Technology Transform Healthcare and Biomedical Sciences?

PMID: 31460519 [PubMed]

PMCID: PMC6711478

EC Pharmacology and Toxicology
Is it a Prime Time for AI-powered Virtual Drug Screening?

PMID: 30215059 [PubMed]

PMCID: PMC6133253

EC Psychology and Psychiatry
Analysis of Evidence for the Combination of Pro-dopamine Regulator (KB220PAM) and Naltrexone to Prevent Opioid Use Disorder Relapse.

PMID: 30417173 [PubMed]

PMCID: PMC6226033

EC Anaesthesia
Arrest Under Anesthesia - What was the Culprit? A Case Report.

PMID: 30264037 [PubMed]

PMCID: PMC6155992

EC Orthopaedics
Distraction Implantation. A New Technique in Total Joint Arthroplasty and Direct Skeletal Attachment.

PMID: 30198026 [PubMed]

PMCID: PMC6124505

EC Pulmonology and Respiratory Medicine
Prevalence and factors associated with self-reported chronic obstructive pulmonary disease among adults aged 40-79: the National Health and Nutrition Examination Survey (NHANES) 2007-2012.

PMID: 30294723 [PubMed]

PMCID: PMC6169793

EC Dental Science
Important Dental Fiber-Reinforced Composite Molding Compound Breakthroughs

PMID: 29285526 [PubMed]

PMCID: PMC5743211

EC Microbiology
Prevalence of Intestinal Parasites Among HIV Infected and HIV Uninfected Patients Treated at the 1o De Maio Health Centre in Maputo, Mozambique

PMID: 29911204 [PubMed]

PMCID: PMC5999047

EC Microbiology
Macrophages and the Viral Dissemination Super Highway

PMID: 26949751 [PubMed]

PMCID: PMC4774560

EC Microbiology
The Microbiome, Antibiotics, and Health of the Pediatric Population.

PMID: 27390782 [PubMed]

PMCID: PMC4933318

EC Microbiology
Reactive Oxygen Species in HIV Infection

PMID: 28580453 [PubMed]

PMCID: PMC5450819

EC Microbiology
A Review of the CD4 T Cell Contribution to Lung Infection, Inflammation and Repair with a Focus on Wheeze and Asthma in the Pediatric Population

PMID: 26280024 [PubMed]

PMCID: PMC4533840

EC Neurology
Identifying Key Symptoms Differentiating Myalgic Encephalomyelitis and Chronic Fatigue Syndrome from Multiple Sclerosis

PMID: 28066845 [PubMed]

PMCID: PMC5214344

EC Pharmacology and Toxicology
Paradigm Shift is the Normal State of Pharmacology

PMID: 28936490 [PubMed]

PMCID: PMC5604476

EC Neurology
Examining those Meeting IOM Criteria Versus IOM Plus Fibromyalgia

PMID: 28713879 [PubMed]

PMCID: PMC5510658

EC Neurology
Unilateral Frontosphenoid Craniosynostosis: Case Report and a Review of the Literature

PMID: 28133641 [PubMed]

PMCID: PMC5267489

EC Ophthalmology
OCT-Angiography for Non-Invasive Monitoring of Neuronal and Vascular Structure in Mouse Retina: Implication for Characterization of Retinal Neurovascular Coupling

PMID: 29333536 [PubMed]

PMCID: PMC5766278

EC Neurology
Longer Duration of Downslope Treadmill Walking Induces Depression of H-Reflexes Measured during Standing and Walking.

PMID: 31032493 [PubMed]

PMCID: PMC6483108

EC Microbiology
Onchocerciasis in Mozambique: An Unknown Condition for Health Professionals.

PMID: 30957099 [PubMed]

PMCID: PMC6448571

EC Nutrition
Food Insecurity among Households with and without Podoconiosis in East and West Gojjam, Ethiopia.

PMID: 30101228 [PubMed]

PMCID: PMC6086333

EC Ophthalmology
REVIEW. +2 to +3 D. Reading Glasses to Prevent Myopia.

PMID: 31080964 [PubMed]

PMCID: PMC6508883

EC Gynaecology
Biomechanical Mapping of the Female Pelvic Floor: Uterine Prolapse Versus Normal Conditions.

PMID: 31093608 [PubMed]

PMCID: PMC6513001

EC Dental Science
Fiber-Reinforced Composites: A Breakthrough in Practical Clinical Applications with Advanced Wear Resistance for Dental Materials.

PMID: 31552397 [PubMed]

PMCID: PMC6758937

EC Microbiology
Neurocysticercosis in Child Bearing Women: An Overlooked Condition in Mozambique and a Potentially Missed Diagnosis in Women Presenting with Eclampsia.

PMID: 31681909 [PubMed]

PMCID: PMC6824723

EC Microbiology
Molecular Detection of Leptospira spp. in Rodents Trapped in the Mozambique Island City, Nampula Province, Mozambique.

PMID: 31681910 [PubMed]

PMCID: PMC6824726

EC Neurology
Endoplasmic Reticulum-Mitochondrial Cross-Talk in Neurodegenerative and Eye Diseases.

PMID: 31528859 [PubMed]

PMCID: PMC6746603

EC Psychology and Psychiatry
Can Chronic Consumption of Caffeine by Increasing D2/D3 Receptors Offer Benefit to Carriers of the DRD2 A1 Allele in Cocaine Abuse?

PMID: 31276119 [PubMed]

PMCID: PMC6604646

EC Anaesthesia
Real Time Locating Systems and sustainability of Perioperative Efficiency of Anesthesiologists.

PMID: 31406965 [PubMed]

PMCID: PMC6690616

EC Pharmacology and Toxicology
A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

PMID: 31517314 [PubMed]

PMCID: PMC6741290

EC Pharmacology and Toxicology
Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

PMID: 31633124 [PubMed]

PMCID: PMC6800226

EC Neurology
Research Volunteers' Attitudes Toward Chronic Fatigue Syndrome and Myalgic Encephalomyelitis.

PMID: 29662969 [PubMed]

PMCID: PMC5898812

EC Pharmacology and Toxicology
Hyperbaric Oxygen Therapy for Alzheimer's Disease.

PMID: 30215058 [PubMed]

PMCID: PMC6133268

News and Events

February Issue Release

We always feel pleasure to share our updates with you all. Here, notifying you that we have successfully released the February issue of respective journals and the latest articles can be viewed on the current issue pages.

Submission Deadline for Upcoming Issue

ECronicon delightfully welcomes all the authors around the globe for effective collaboration with an article submission for the upcoming issue of respective journals. Submissions are accepted on/before February 17, 2023.

Certificate of Publication

ECronicon honors with a "Publication Certificate" to the corresponding author by including the names of co-authors as a token of appreciation for publishing the work with our respective journals.

Best Article of the Issue

Editors of respective journals will always be very much interested in electing one Best Article after each issue release. The authors of the selected article will be honored with a "Best Article of the Issue" certificate.

Certifying for Review

ECronicon certifies the Editors for their first review done towards the assigned article of the respective journals.

Latest Articles

The latest articles will be updated immediately on the articles in press page of the respective journals.