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Introduction
Dental caries, ubiquitously acknowledged as the most widespread noncommunicable disease, afflicts over a third of the global population, with untreated cases surpassing 2 billion individuals worldwide (1). This pathology, predominantly caused by the bacterial agent Streptococcus mutans, leads to the degradation of dental tissues due to acidic by-products from bacterial metabolism (2).
S. mutans, adept at colonizing the dental biofilm, metabolizes free sugars, particularly sucrose, from food and beverages into acids, progressively leading to tooth enamel demineralization (3). While mechanical removal of biofilm via toothbrushing is the cornerstone in caries management, alternative strategies have garnered research interest for comprehensive caries control (4).
Probiotics, defined as live microorganisms which, when administered in adequate amounts, confer a health benefit to the host (5), have emerged as a potential adjunct in dental health strategies. The spectrum of probiotics spans various genera, including but not limited to Lactobacillus, Pediococcus, Propionibacterium, and Bifidobacterium. These microorganisms, having established beneficial roles in diverse health conditions such as gut inflammation, hypercholesterolemia, and urogenital infections (6), are now being investigated for their efficacy in the oral health domain.
Clinical trials in dentistry have frequently employed probiotics from Lactobacillus, Bifido- bacterium, Streptococci, and Bacillus genera (7). While the precise impact of these probiotics on dental caries is yet to be fully elucidated, it is hypothesized that they contribute to pathogenic biofilm disruption and modulate host defense mechanisms, including mucosal immune defense and macrophage activity (8). For efficacious caries prevention, probiotics must not only adhere to and integrate into dental biofilms but also exhibit competitive attributes against cariogenic bacteria and a low-acid-producing metabolic profile (9).
Identifying an optimal vehicle for probiotic delivery remains an area of active research. Dairy products like yogurt, milk, and cheese are tradi- tionally deemed suitable carriers. However, the widespread appeal and acceptance of ice cream, particularly among pediatric populations, present it as a novel and potentially effective medium for probiotic delivery (10,11). This systematic review, therefore, aims to critically evaluate the impact of probiotic-enriched ice cream on the reduction of Streptococcus mutans bacterial counts, a key indicator in dental caries prevention.
Material and methods
Protocol and registration
This systematic review follows all recommendations proposed by the PRISMA - Preferred Reporting Items for Systematic Reviews and Meta-Analyses (12). The authors have registered this research on the PROSPERO (International Prospective Register of Systematic Reviews) platform under the CRD42022371298. The availability data may be request to the corresponding author (PHSS).
Eligibility criteria
Clinical trials in which subjects were fed an ice cream enriched with a probiotic diet were included to analyze the salivary count of Strepto- coccus mutans. There was no restriction on the probiotic used to prepare the ice cream, year, or publication language. Other study designs than original articles and research without ice cream were excluded.
Information source and search
The search strategy construction process was based on the PICO question: P (population) - syste- matically healthy individuals; I (intervention) - ice cream enriched with probiotic diet; C (comparison)- ice cream diet without probiotics; O (outcome)- salivary S. mutans count. The databases used for the search were Scopus, Web of Science, Embase, Pubmed, Medline, and Science Direct, conducted between October 25 and 30, 2022.
Thus, the search strategy was: (teeth or tooth or “dental caries” or “dental caries activity test” or “dental decay” or “decay, dental” “carious lesion”) and (probiotic or “dietary supplements” or synbio- tics or prebiotics or “polysaccharides, bacterial” or lactobacillus or icrecream or “ice-cream” or “ice cream” or “cream, ice”) and (“colony forming units” or antibacterial or saliva or “saliva samples”).
Selection of evidence sources
The selection of the studies included in this review was performed by two independent blinded reviewers (MJFC and PHSS) using the Rayyan online platform - rayyan.qcri.org (13). The first step relied on eliminating duplicates and reading the titles, abstracts, and the whole paper. Incon- sistencies were resolved in an agreement between the two examiners. The inter-examiner agreement was assessed by Cohen's Kappa test with a value of 0.92 (CI=0.91-0.93).
Data charting process
The information extracted from the articles (authors, year of publication, country of publica- tion, sample, sample groups, time of analysis, probiotic, test used for salivary quantification of S.mutans, and main results) was grouped, filled in, and checked by the two independent researchers (MJFC and PHSS) to examine possible inconsistencies and ensure data accuracy using Microsoft Office Excel 2016.
Critical appraisal of individual sources of evidence
The risk of bias analysis was performed using the RoB2 platform developed by Cochrane. The individual analysis of included articles was checked against six domains that briefly consider selection bias, performance bias, detection bias, attrition bias, reporting bias, and any other condition found. All articles included in this analysis were checked by two independent reviewers (MJFC and PHSS), and inconsistencies were resolved in an agreement between the two reviewers.
Regarding the quality of the scientific evidence, we used the GRADE (Grades of Recommendation, Assessment, Development and Evaluation) that evaluates the certainty assessment based on study design, risk of bias, inconsistency, indirect evidence, imprecision, and other considerations. The information was consolidated in the online software "GRADEPRO" by one of the researchers trained to manipulate the platform (MJFC) (14).
Synthesis of data
Data were analyzed using Revman 5.4.1. Data from the included studies were ordinal for the "Colony Formation Unit" outcome measure. Risk differences were used as an effect measure with 95% confidence intervals and a random effects model. We assessed heterogeneity using I² statistics. Data from some follow-up periods (1 hour, 7, 18, 30, 90, and 180 days) were included for the outcome.
Results
We obtained 63 studies from all databases, in which 18 were duplicated. After screening, we included 6 articles in the final sample. The Figure 1 show the detailed selection process following PRISMA flow diagram.
We excluded seven studies that met eligibility criteria: six studies presented only in vitro approach (14,15,16,17,18,19), one study use yogurt rather than ice-cream as “vehicle” (20).
Studies characteristics
According to the obtained results, probiotic containing ice cream reduced S. mutans salivary load (CFU) in all the primary studies of this syste- matic review. The included articles used Bifidobac- terium lactis Bb-12 (n=5; 83.3%) (21,22,23,24,25) combined or not with Lactobacillus acidophilus La-5 (n=3; 50.0%) (22,23,24), while only one study did not present the used probiotic (26). The studies were conducted in India (n=5; 83.3%) (22,23,24,25,26) or Türkiye (n=1; 16.7%) (21). In the selected studies, the sample range from 20 up to 60 participants, with a media of 37 individuals. The evaluation time ranged from 1 hour (26) up to 180 days (23). Five studies were published in the years 2010-2019 (n=83.3%) (22,23,24,26) and one was conducted before 2010 (21). The detailed information can be consulted in Table 1.
Risk bias in studies
The studies were very similar in the classification of the proposed tool. Four studies presented low risk of bias for all domains (23,24,25,26). Other studies did not present all results, as media and standard deviation (21,22). According to the proposed protocol and the criteria previously explained in the material and methods section (2.2), all studies were classified as having a low risk of bias. Detailed information of each study can be visualized in Table 2.
In this study, we also evaluated the evidence quality using the GRADE. All the 6 included studies were Randomized Clinical Trials (RCT), with non-severe problems about risk of bias, inconsistency, indirect evidence, and imprecision. Thus, we consider the evidence quality certainty as high.
Results of syntheses descriptive analysis
We included in the meta-analysis four studies that reported the CFU (media and standard devia- tion) in different periods. The difference in the number of studies in each follow-up is attributed to whether the outcome was reported by the authors. The follow-up of 18, 30, 90, and 180 days presented only one study each.
Meta-analysis
The risk difference for the comparison between ice-cream containing probiotics and ice-cream without probiotics for the 1-hour follow-up period was -50.01 with 95% CI between (-158.66, 58.64), with no statistically significant difference (p=0.370). The 7-days follow-up risk difference was -70.77 with CI between (-118.42,-23.12), with statistically significant difference (p=0.004). Overall heterogeneity was high with I²>75%. Taking all the results together, the risk difference was -40.85 (-59.77, -21.93), with a statistically significant difference (p<0.0001) (Figure 2).
Table 1 Analysis of articles included in the systematic review.
| Study | Local | Sample | Groups | Time | Probiotic | Test |
|---|---|---|---|---|---|---|
| Ashwin24 | India | 60 children (6-12 years) | G1: probiotic (n=30); G2: placebo (n=30) | 7, 30, and 180 days | Bifidobacterium lactis Bb-12; Lactobacillus acidophilus La-5 | CFU |
| Mahantesha28 | India | 50 children (6-12 years) | G1: probiotics in ice-cream (n=25); G2: probiotics in drink (n=25) | 7 and 90 days | Ice-cream: Bifidobacterium lactis Bb-12; Lactobacillus acidophilus La-5 Drink: Lactobacillus casei | CFU |
| Chinnappa28 | India | 20 children (12-14 years) | G1: probiotic (n=10); G2: placebo (n=10) | 1 hour and 7 days | - | CFU |
| Singh28 | India | 40 children (12-14 years) | G1: probiotic (n=20); G2: placebo (n=20) | 7 and 14 days | Bifidobacterium lactis Bb-12; Lactobacillus acidophilus La-5 | CFU |
| Çaglar25 | Türkiye | 24 individuals | G1: probiotic (n=12); G2: placebo (n=12) | 10 and 20 days | Bifidobacterium lactis Bb-12 | CFU |
| Nagarajappa27 | India | 30 individuals | G1: probiotic (n=15); G2: placebo (n=15) | 1 hour and 18 days | Bifidobacterium lactis Bb-12 | CFU |
CFU: Colony Forming Unit.
Table 2 Risk of bias assessment.
| Study | Domain 1 | Domain 2 | Domain 3 | Domain 4 | Domain 5 | Domain 6 |
|---|---|---|---|---|---|---|
| Ashwin et al.²⁴ | - | - | - | - | - | - |
| Çaglar et al.2⁵ | - | - | - | ? | - | - |
| Chinnappa et al.²⁹ | - | - | - | - | - | - |
| Mahantesha et al.2⁶ | - | - | - | - | - | - |
| Nagarajappa et al.2⁷ | - | - | - | - | - | - |
| Singh et al.2⁸ | - | - | - | ? | - | - |
+: High risk ok bias; -: Low risk of bias; ?: Not founded. Domain 1: risk of bias arising from the randomization process; Domain 2: effect of assignment to intervention; Domain 3: effect of adhering to intervention; Domain 4: missing outcome data; Domain 5: risk of bias in measurement of the outcome; Domain 6: risk of bias in selection of the reported result.
Discussion
The strategic incorporation of probiotics in dental caries management epitomizes a novel paradigm in contemporary oral health research. This systematic review meticulously scrutinizes the efficacy of probiotic-fortified ice cream in attenuating the salivary concentrations of Streptococcus mutans. Notably, this study reveals a substantive decrement in S. mutans load, sustained for up to 7 days subsequent to the ingestion of probiotic- infused ice cream.
The realm of functional foods, particularly dairy-based products, has been recognized as a conducive matrix for probiotic delivery (10, 11, 27). Expanding upon this notion, this study delineates the potential role of ice cream as a palatable and efficacious probiotic carrier. This is particularly relevant in pediatric contexts, where acceptabi- lity and compliance with health interventions are paramount. The comparative analysis undertaken by Mahantesha et al. (2015) (24) accentuates the superiority of ice cream, relative to other probiotic vehicles, in mitigating S. mutans levels.
Delving deeper, the research by Çaglar et al. (2008) (21) delineates a significant reduction in S. mutans in young adults following a regimen of B. lactis Bb-12 enriched ice cream. This not only illuminates the strain-specific efficacy in oral health interventions but also highlights the nonexistence of detrimental effects. The researchers, however, astutely acknowledge the constraints of their study, including limited sample size and the transient nature of salivary mutans streptococci as an inter- mediate marker for caries progression.
In a similar vein, the incorporation of bifido- bacteria in a chocolate-based ice cream was observed to reduce salivary S. mutans and lactobacilli by 26.8% over an 18-day period in adult participants (25). The intrinsic attributes of ice cream, enriched with casein, calcium, and phosphorus, coupled with its distinctive textural and sensory characteristics, amplify its suitability as a probiotic conduit (9).
Moreover, this review elucidates a consistent reduction in S. mutans across diverse pediatric studies, with the duration of probiotic consumption emerging as a critical determinant in the efficacy of these interventions (22,23,24,25,26). The probiotic- induced biofilm formation acts as a defensive barrier, mitigating cariogenic microorganisms and suggesting a viable preventive approach in pediatric dentistry (11,28).
Diverse concentrations of B. lactis Bb-12 and L. acidophilus La-5 were employed across the studies under review (22,23,24,25,26), each strain exhibiting unique properties conducive to oral health. B. lactis Bb-12 is renowned for its adhesion capabilities, crucial for effective colonization in the oral cavity (29), while L. acidophilus La-5 is distinguished by its bacteriocin production, instrumental in targeting S. mutans within the oral biofilm (30, 31). Yet, the heterogeneity in administration protocols underscores the necessity for standardized methodologies in future research endeavors.
A critical limitation of this review is the exclusion of a sensitivity analysis to appraise the heterogeneity across the included studies, a facet that could potentially influence the interpre- tative breadth of our findings (32). The variations in enrichment protocols and follow-up durations further necessitate a nuanced interpretation of the results. Despite these challenges, this review represents an inaugural comprehensive synthesis of randomized controlled trials exploring the efficacy of probiotic-enriched ice cream in diminis- hing S. mutans counts, a pivotal step in dental caries prevention.
It is imperative to underscore that while certain probiotics like B. lactis Bb-12 exhibit resilience in low pH environments, indicative of their potential efficacy in the oral milieu (29), their acidogenic propensity in symbiosis with S. mutans raises concerns regarding enamel demineralization in complex biofilm matrices (16). Consequently, future investigations must be directed towards unraveling the in situ interactions between probiotics and oral biofilms, identifying optimal probiotic strains and dosages, and elucidating their long-term implications on oral health, particularly concerning enamel integrity and the demineralization-remineralization dynamic (33).
Conclusions
Probiotic-enriched ice cream, particularly containing strains such as B. lactis Bb-12 and L. acidophilus La-5, significantly reduces salivary levels of Streptococcus mutans. The uniformity in the reduction of S. mutans across different study populations underlines the potential of probiotic ice cream as a universally acceptable and effec- tive medium.
Author contribution statement
Conception or design: P.H.S.S., R.S.A., R.O.C.,S.I.C.S., L.R.A.L. and M.J.F.C.
Acquisition, analysis, or interpretation of data: P.H.S.S., R.S.A., R.O.C., S.I.C.S., L.R.A.L. and M.J.F.C.
Drafting the work or reviewing it critically for important intellectual content: S.I.C.S., L.R.A.L. and M.J.F.C.
Final approval of the version to be published:P.H.S.S. and M.J.F.C.
