A new approach for activation of the kiwifruit cysteine protease for usage in in-vitro testing.

Actinidin (Act d 1), a highly abundant cysteine protease from kiwifruit, is one of the major contributors to the development of kiwifruit allergy. Many studies have focused on the optimization of Act d 1 purification and its role in the development of food allergies. Testing on cell culture monolayers is a common step in the elucidation of food allergen sensitization. In the case of cysteine proteases, an additional activation step with L-cysteine is required before the testing. Hence, we aimed to evaluate whether L-cysteine already present in commonly used cell culture media would suffice for Act d 1 activation. Successfully activated Act d 1 (98.1% of proteolytic activity, as compared to L-cysteine activated Act d 1) was further tested in two commonly used 2D model systems (Caco-2 and HEK293 cells) to evaluate its role on the mRNA expression of cytokines involved in the innate immunity (IL-1ß, IL-6, TNFα, TSLP). Furthermore, the contribution of Act d 1 in the promotion of inflammation through regulation of inducible nitric oxide synthase (iNOS) mRNA expression was also examined. These results demonstrate that activation of cysteine proteases can be achieved without previous enzyme incubation in L-cysteine -containing solution. Act d 1 incubated in cell culture medium was able to modulate gene expression of pro-inflammatory cytokines when tested on two model systems of the epithelial barrier.

Putative mechanisms of kiwifruit on maintenance of normal gastrointestinal function.

Kiwifruits are recognized as providing relief from constipation and symptoms of constipation-predominant irritable bowel syndrome (IBS-C). However, the underlying mechanisms, specifically in regards to gastrointestinal transit time and motility, are still not completely understood. This review provides an overview on the physiological and pathophysiological processes underlying constipation and IBS-C, the composition of kiwifruit, and recent advances in the research of kiwifruit and abdominal comfort. In addition, gaps in the research are highlighted and scientific studies of other foods with known effects on the gastrointestinal tract are consulted to find likely mechanisms of action. While the effects of kiwifruit fiber are well documented, observed increases in gastrointestinal motility caused by kiwifruit are not fully characterized. There are a number of identified mechanisms that may be activated by kiwifruit compounds, such as the induction of motility via protease-activated signaling, modulation of microflora, changes in colonic methane status, bile flux, or mediation of inflammatory processes.

Effects of Ultrasound Assisted Extraction in Conjugation with Aid of Actinidin on the Molecular and Physicochemical Properties of Bovine Hide Gelatin.

Actinidin was used to pretreat the bovine hide and ultrasonic wave (53 kHz and 500 W) was used for the time durations of 2, 4 and 6 h at 60 °C to extract gelatin samples (UA2, UA4 and UA6, respectively). Control (UAC) gelatin was extracted using ultrasound for 6 h at 60 °C without enzyme pretreatment. There was significant (p < 0.05) increase in gelatin yield as the time duration of ultrasound treatment increased with UA6 giving the highest yield of 19.65%. Gel strength and viscosity of UAC and UA6 extracted gelatin samples were 627.53 and 502.16 g and 16.33 and 15.60 mPa.s, respectively. Longer duration of ultrasound treatment increased amino acids content of the extracted gelatin and UAC exhibited the highest content of amino acids. Progressive degradation of polypeptide chains was observed in the protein pattern of the extracted gelatin as the time duration of ultrasound extraction increased. Fourier transform infrared (FTIR) spectroscopy depicted loss of molecular order and degradation in UA6. Scanning electron microscopy (SEM) revealed protein aggregation and network formation in the gelatin samples with increasing time of ultrasound treatment. The study indicated that ultrasound assisted gelatin extraction using actinidin exhibited high yield with good quality gelatin.

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions.

BACKGROUND: The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). METHODS: Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. RESULTS: Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 µg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 µg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. CONCLUSION: Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. GENERAL SIGNIFICANCE: In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.

Conformational mobility of active and E-64-inhibited actinidin.

BACKGROUND: Actinidin, a protease from kiwifruit, belongs to the C1 family of cysteine proteases. Cysteine proteases were found to be involved in many disease states and are valid therapeutic targets. Actinidin has a wide pH activity range and wide substrate specificity, which makes it a good model system for studying enzyme-substrate interactions. METHODS: The influence of inhibitor (E-64) binding on the conformation of actinidin was examined by 2D PAGE, circular dichroism (CD) spectroscopy, hydrophobic ligand binding assay, and molecular dynamics simulations. RESULTS: Significant differences were observed in electrophoretic mobility of proteolytically active and E-64-inhibited actinidin. CD spectrometry and hydrophobic ligand binding assay revealed a difference in conformation between active and inhibited actinidin. Molecular dynamics simulations showed that a loop defined by amino-acid residues 88-104 had greater conformational mobility in the inhibited enzyme than in the active one. During MD simulations, the covalently bound inhibitor was found to change its conformation from extended to folded, with the guanidino moiety approaching the carboxylate. CONCLUSIONS: Conformational mobility of actinidin changes upon binding of the inhibitor, leading to a sequence of events that enables water and ions to protrude into a newly formed cavity of the inhibited enzyme. Drastic conformational mobility of E-64, a common inhibitor of cysteine proteases found in many crystal structures stored in PDB, was also observed. GENERAL SIGNIFICANCE: The analysis of structural changes which occur upon binding of an inhibitor to a cysteine protease provides a valuable starting point for the future design of therapeutic agents.

Active actinidin retains function upon gastro-intestinal digestion and is more thermostable than the E-64-inhibited counterpart.

BACKGROUND: Actinidin is a cysteine protease and major allergen from kiwi fruit. When purified under specific native conditions, actinidin preparations from fresh kiwi fruit contain both an active and inactive form of this enzyme. In this study, biochemical and immunological properties upon simulated gastro-intestinal digestion, as well as thermal stability, were investigated for both active and E-64-inhibited actinidin. RESULTS: Active actinidin retained its primary structure and proteolytic activity after 2 h of simulated gastric digestion, followed by 2 h of intestinal digestion, as assessed by SDS-PAGE, zymography and mass spectroscopy. Immunological reactivity of active actinidin was also preserved, as tested by immunoelectrophoresis. The E-64 inhibited actinidin was fully degraded after 1 h of pepsin treatment. Differential scanning calorimetry showed that active actinidin has one transition maximum temperature (Tm ) at 73.9°C, whereas in the E-64-actinidin complex the two actinidin domains unfolded independently, with the first domain having a Tm value of only 61°C. CONCLUSION: Active actinidin is capable of reaching the intestinal mucosa in a proteolytically active and immunogenic state. Inhibitor binding induces changes in the actinidin molecule that go beyond inhibition of proteolytic activity, also influencing the digestion stability and Tm values of actinidin, features important in the characterisation of food allergens.

Effects of Green and Gold Kiwifruit Varieties on Antioxidant Neuroprotective Potential in Pigs as a Model for Human Adults.

Kiwifruit (KF) has shown neuroprotective potential in cell-based and rodent models by augmenting the capacity of endogenous antioxidant systems. This study aimed to determine whether KF consumption modulates the antioxidant capacity of plasma and brain tissue in growing pigs. Eighteen male pigs were divided equally into three groups: (1) bread, (2) bread + Actinidia deliciosa cv. 'Hayward' (green-fleshed), and (3) bread + A. chinensis cv. 'Hort16A' (yellow-fleshed). Following consumption of the diets for eight days, plasma and brain tissue (brain stem, corpus striatum, hippocampus, and prefrontal cortex) were collected and measured for biomarkers of antioxidant capacity, enzyme activity, and protein expression assessments. Green KF significantly increased ferric-reducing antioxidant potential (FRAP) in plasma and all brain regions compared with the bread-only diet. Gold KF increased plasma ascorbate concentration and trended towards reducing acetylcholinesterase activity in the brain compared with the bread-only diet. Pearson correlation analysis revealed a significant positive correlation between FRAP in the brain stem, prefrontal cortex, and hippocampus with the total polyphenol concentration of dietary interventions. These findings provide exploratory evidence for the benefits of KF constituents in augmenting the brain's antioxidant capacity that may support neurological homeostasis during oxidative stress.

In-vitro and in-silico analyses of the thrombolytic potential of green kiwifruit.

Cardiovascular diseases (CVDs), mainly caused by thrombosis complications, are the leading cause of mortality worldwide, making the development of alternative treatments highly desirable. In this study, the thrombolytic potential of green kiwifruit (Actinidia deliciosa cultivar Hayward) was assessed using in-vitro and in-silico approaches. The crude green kiwifruit extract demonstrated the ability to reduce blood clots significantly by 73.0 ± 1.12% (P < 0.01) within 6 h, with rapid degradation of Aα and Bß fibrin chains followed by the γ chain in fibrinolytic assays. Molecular docking revealed six favorable conformations for the kiwifruit enzyme actinidin (ADHact) and fibrin chains, supported by spontaneous binding energies and distances. Moreover, molecular dynamics simulation confirmed the binding stability of the complexes of these conformations, as indicated by the stable binding affinity, high number of hydrogen bonds, and consistent distances between the catalytic residue Cys25 of ADHact and the peptide bond. The better overall binding affinity of ADHact to fibrin chains Aα and Bß may contribute to their faster degradation, supporting the fibrinolytic results. In conclusion, this study demonstrated the thrombolytic potential of the green kiwifruit-derived enzyme and highlighted its potential role as a natural plant-based prophylactic and therapeutic agent for CVDs.

Understanding the Molecular Basis for Enhanced Glutenase Activity of Actinidin using Structural Bioinformatics.

BACKGROUND: Management of gluten intolerance is currently possible only by consumption of a gluten-free diet (GFD) for a lifetime. The scientific community has been searching for alternatives to GFD, like the inclusion of natural proteases with meals or pre-treatment of gluten-containing foods with glutenases. Actinidin from kiwifruit has shown considerable promise in digesting immunogenic gliadin peptides compared to other plant-derived cysteine proteases. METHODS: In this study, we aimed to understand the structural basis for the elevated protease action of actinidin against gliadin peptides by using an in silico approach. RESULTS: Docking experiments revealed key differences between the binding of gliadin peptide to actinidin and papain, which may be responsible for their differential digestive action. CONCLUSION: Sequence comparison of different plant cysteine proteases highlights amino acid residues surrounding the active site pocket of actinidin that are unique to this molecule and hence likely to contribute to its digestive properties.

Influence of Actinidin-Induced Hydrolysis on the Functional Properties of Milk Protein and Whey Protein Concentrates.

The main aim of the study was to establish the impact of limited proteolysis by actinidin on the functionality of selected milk protein systems. The plant protease actinidin was used to produce hydrolysates (MPHs) from milk protein concentrate (MPC) and whey protein concentrate (WPC) to 0, 5, 10 or 15% of the degree of hydrolysis (DH) at an enzyme-to-substrate ratio of 1:100 (5.21 units of actinidin activity g-1 of protein). The functionalities assessed included solubility, heat stability, emulsification and foaming properties. In general, significant changes in the functionalities of MPH were associated with the extent of hydrolysis. Solubility of hydrolysates increased with increasing %DH, with WPC showing about 97% solubility at 15% DH. Emulsifying properties were negatively affected by hydrolysis, whereas heat stability was improved in the case of WPC (~25% of heat stability increased with an increase in DH to 15%). Hydrolysates from both WPC and MPC had improved foaming properties in comparison to unhydrolysed controls. These results were also supported by changes in the FTIR spectra. Further adjustment of hydrolysis parameters, processing conditions and pH control could be a promising approach to manipulate selected functionalities of MPHs obtained using actinidin.

Milk Protein Hydrolysis by Actinidin-Kinetic and Thermodynamic Characterisation and Comparison to Bromelain and Papain.

Plant proteases, including actinidin, papain and bromelain, have been widely used in the food industry but with limited application in dairy systems. This research aimed to establish and compare operational parameters (kinetics, temperature, enzyme type, time and thermodynamics) relevant to the applications of these enzymes in the hydrolysis of whey protein isolates (WPI), whey protein concentrates (WPC) or milk protein concentrates (MPC). The degree of hydrolysis (DH) increased with the rise in temperature, and the maximum DH was achieved at 60 °C for all three dairy systems. The addition of papain resulted in a greater %DH of whey proteins in comparison to bromelain. The cleavage of proteins was clearly time-dependent (p < 0.05), while the pH did not change significantly (p > 0.05) during this time. PAGE analysis revealed that all three enzymes mainly acted on α-lactalbumin and αs-casein in WPI and MPC, respectively. Kinetic parameters from the Lineweaver-Burk plot at 60 °C using WPC and MPC as a substrate varied widely, establishing that WPC hydrolysis was characterised by a lower KM, higher kcat, kcat/KM and Vmax compared to MPC in the case of all three enzymes. The difference in kcat/KM values amongst all enzymes (actinidin > papain > bromelain) indicated the difference in the strength of substrate binding sites. The thermodynamic parameters of these enzymes with MPC and WPC were also determined at a temperature range of 15-60 °C, and the results indicate the potential application of papain and actinidin in the dairy industry.

An Easy and Cheap Kiwi-Based Preparation as Vegetable Milk Coagulant: Preliminary Study at the Laboratory Scale.

In the present study, a kiwifruit aqueous extract was developed and used as a coagulant enzyme in cheesemaking. In detail, polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the presence of actinidin, the kiwifruit enzyme involved in κ-casein hydrolysis, in different tissues (pulp, peel, and whole fruit) of ripe and unripe kiwifruits. Data revealed the presence of the enzyme both in the peel and in the pulp of the fruit. Although the aqueous extract obtained from the kiwifruit peel was able to hydrolyze semi-skimmed milk, it did not break down κ-casein. The aqueous extract obtained from the pulp showed a hydrolytic activity toward both κ-casein and semi-skimmed milk. The values for milk-clotting and proteolytic activity of the kiwifruit pulp extract were evaluated at different temperatures and pH parameters in order to obtain a high value of the MCA/PA ratio; we found that a temperature of 40 °C in combination with a pH value of 5.5 allowed us to obtain the best performance. In addition, the data revealed a higher hydrolytic activity of the enzymatic preparation from ripe kiwifruits than that from unripe ones, suggesting the use of the extract from pulp of ripe kiwifruits in the laboratory-scale cheesemaking. The data showed that 3% (v/v) of the ripe kiwifruit pulp extract determined a curd yield of 20.27%, comparable to chymosin yield. In conclusion, the extraction procedure for kiwifruit aqueous extract proposed in the present study was shown to be a fast, cheap, chemical-free, and ecofriendly technology as a plant coagulant for cheese manufacturing.

Actinidin in Green and SunGold Kiwifruit Improves Digestion of Alternative Proteins-An In Vitro Investigation.

Both Hayward (green) and SunGold (gold) kiwifruit varieties contain a proteolytic enzyme, actinidin, that has been reported to enhance the upper tract digestion of animal proteins. Unlike the other gold varieties, which do not contain any actinidin, the SunGold variety contains significantly higher actinidin activity, but its activity is still much lower than that present in the green (Hayward) fruit. The objective of this study was to determine the effectiveness of actinidin in Hayward and SunGold kiwifruit in digesting alternative proteins, including pea protein, almonds, tofu, and quinoa. The protein sources were digested using a three-stage in vitro oral-gastro-small intestinal digestion model. The findings showed that both kiwifruit extracts enhanced the breakdown (observed through SDS-PAGE) for all the studied protein sources, particularly during gastric digestion, possibly due to higher actinidin activity at gastric pH. The increase in the rate of protein breakdown was probably due to the broader specificity of actinidin compared to pepsin. For many protein sources, most of the intact proteins disappeared within the first few minutes of gastric digestion with added kiwifruit extract. Green kiwifruit extract, due to its higher actinidin activity, had a higher effect on protein breakdown than the SunGold extract. However, for some proteins and under certain digestion conditions, SunGold extract resulted in higher protein breakdown. The latter, in the absence of any digestive enzymes, also led to some protein breakdown during the small intestinal digestion phase, which was not the case for the green kiwifruit extract. The green kiwifruit extract led to the greater breakdown of polypeptide chains of Pru-du 6, a major allergen in almonds. The results, for the first time, suggest that both Hayward and SunGold kiwifruit can lead to improved breakdown and digestion of alternative proteins when consumed as part of a meal; and therefore, have the potential to be used as a digestive aid in population groups looking to achieve faster and greater protein digestion such as athletes, elderly and people with the impaired digestive system.

Actinidin reduces gluten-derived immunogenic peptides reaching the small intestine in an in vitro semi-dynamic gastrointestinal tract digestion model.

Actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), has been identified as a potential enzyme to hydrolyse gluten within the lumen of the gastrointestinal tract (GIT). The present study aimed to further evaluate the effect of purified actinidin sourced from green kiwifruit on the digestion of gluten and the release of immunogenic peptides during GIT digestion using an in vitro semi-dynamic GIT digestion model. Purified gluten was digested for 180 min with or without actinidin and subsequently analysed for free amino groups (o-phthaldialdehyde) to determine the degree of hydrolysis (DH), gluten R5 epitopes (ELISA), and peptide profiles (mass spectrometry). Strong interactions were observed between treatment (GIT digestion with or without actinidin) and digestion time for the DH of gluten (P < 0.01), amount of free amino groups released into the small intestine (P < 0.01), and amount of gluten epitopes present in the small intestine (P < 0.001). The rate of increase of DH of gluten and the amount of R5 epitopes present in the small intestine during the first 30 min of GIT digestion with actinidin was 0.3%/min and 4.8 ng/g of gluten respectively, whereas it was 0.01%/min and 60.9 ng/g of gluten respectively without actinidin. These results were corroborated by untargeted peptidomics, with a 1.5-fold lower number of known immunogenic epitopes reaching the small intestine at 30 min of GIT digestion when actinidin was present compared to the control. Present results demonstrate that actinidin enhanced the rate of proteolysis of gluten and reduced the number of immunogenic gluten epitopes reaching the small intestine during simulated semi-dynamic GIT digestion.

The Effect of Kiwifruit Therapeutics in the Treatment of Diabetic Foot Ulcer.

Diabetes mellitus is considered a silent disease with possible late chronic complications such as diabetic foot ulcer. This condition is managed by surgical debridement. To improve surgical outcome, some surgeons use proteolytic agents after surgery. Kiwifruit contains a type of proteolytic enzyme called actinidin that may play a role in the treatment of such complication. In the current study, we evaluate the role of kiwifruit extract in the treatment of diabetic foot ulcer. Eighteen diabetic foot ulcer patients were included in a randomized, double-blind clinical trial. The patients were divided randomly to control and experimental groups. Patients in the control group underwent daily wound dressing using base ointment (Eucerin). In the experimental group, we added kiwifruit extract to the standard wound dressing. Clinical data including general appearance of wound (according to recorded photographs before and after medical intervention) were analyzed using SPSS version 22. The mean wound area of the experimental group was significantly less than in the control group (P = .005) after 4 weeks of treatment. Comparison of the average of size difference, before and after the treatment in the experimental group and the control group, shows that kiwifruit can have a good impact on wound healing (P = .0001). In patients with diabetic foot ulcer, wound dressing using kiwifruit extract may help reduce time of treatment and may replace surgical debridement for some selected cases.

The kiwifruit enzyme actinidin enhances the hydrolysis of gluten proteins during simulated gastrointestinal digestion.

This study investigated the effect of actinidin, a cysteine protease in kiwifruit, on the hydrolysis of gluten proteins and digestion-resistant gluten peptides (synthetic 33-mer peptide and pentapeptide epitopes) under static simulated gastrointestinal conditions. Actinidin efficacy in hydrolysing gliadin was compared with that of other gluten-degrading enzymes. Actinidin hydrolysed usually resistant peptide bonds adjacent to proline residues in the 33-mer peptide. The gastric degree of hydrolysis of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05), whereas the pentapeptide epitopes hydrolysis was influenced only by the actinidin concentration (P < 0.05). The rate of gastric degree of hydrolysis of gliadin was greater (P < 0.05) by actinidin (0.8%/min) when compared to papain, bromelain, and one commercial enzyme (on average 0.4%/min), while all exogenous enzymes were able to hydrolyse the pentapeptide epitopes effectively. Actinidin is able to hydrolyse gluten proteins under simulated gastric conditions.

Nutraceutical and Technological Properties of Buffalo and Sheep Cheese Produced by the Addition of Kiwi Juice as a Coagulant.

Kiwifruit is an interesting alternative to chymosin for milk coagulation. Although the clotting properties of actinidin (the proteolytic agent present in kiwi) have been widely investigated, little is known about the nutraceutical and organoleptic effects of kiwifruit on the characteristics of cheese. We investigated kiwifruit pulp, compared to calf rennet, in cheesemaking using sheep and buffalo milk. Although the kiwifruit extract showed a longer coagulation and syneresis time than calf rennet, it could nevertheless be exploited as a plant coagulant due to its positive effect on the nutraceutical properties. In fact, the sheep and buffalo cheese were higher in polyphenols and phytosterols than the cheese obtained using calf rennet. In addition, the nutraceutical properties were enhanced, with just a slight effect on the aroma of the cheese.

Shelf-life extension of whole shrimp using an active coating containing fish skin gelatin hydrolysates produced by a natural protease.

This study was focused on shelf-life extension of whole shrimp (Penaeus merguiensis) using an active coating containing gelatin hydrolysates. Gelatin extracted from Scomberomorus commerson skin was hydrolyzed using actinidin extracted from kiwifruit. Some important physicochemical characteristics of fish skin gelatin including viscosity, gelling and melting points, and temperatures were examined. The whole shrimp was coated with four coating agents including fish skin gelatin (FG), commercial gelatin (CG), fish skin gelatin containing 1 mg/ml fish gelatin hydrolysates (FG + GH), and commercial bovine gelatin containing 1 mg/ml fish gelatin hydrolysates (CG + GH). Chemical, microbial, and sensorial properties of samples were monitored for 12 days at 4°C with 3-day intervals (0-12 days). The pH value of samples coated with FG + GH and CG + GH showed the lowest changes during 12 days of storage (1.68 ± 0.00 and 1.70 ± 0.09, respectively). The free fatty acid content (FFA), total volatile base nitrogen (TVB-N), lipid oxidation, and carbonyl content of samples coated with FG + GH and CG + GH were significantly lower than that of control, CG, and FG samples. The results of this study showed that the gelatin hydrolysates could be used as a preservative costing agent for whole shrimp.

The effect of whole carcase medium voltage electrical stimulation, tenderstretching and longissimus infusion with actinidin on alpaca meat quality.

The effect on alpaca meat quality from applying medium voltage electrical stimulation (ES) in combination with tenderstretching (TS; pubic symphysis suspended) to whole carcases was investigated, along with the effect of actinidin infusion on alpaca longissimus (LTL) quality. Carcases (n = 36) were allocated to either no ES + achilles hung; or ES + TS. The left- and right-hand side LTL of each carcase was allocated to one of three infusion treatments; no infusion (control), infusion with water or infusion with enzyme. Processing treatments reduced LTL and semimembranosus shear force without negatively impacting colour or oxidation traits. Infusion with enzyme reduced LTL shear force relative to control and water treatments but resulted in reduced consumer acceptance. The use of TS with ES in commercial alpaca processing is supported. There was no advantage to infusing alpaca LTL with actinidin as results indicate a net negative effect on consumer acceptance of this novel meat.

Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets.

Background: Oral microbiota has been linked to both health and diseases. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested. Results: Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed amplicon sequence variants (254 ± 53 in saliva and 175 ± 37 in tongue; P = 8.9e-7, Kruskal-Wallis test) and Shannon index (6.0 ± 0.4 in saliva and 5.4 ± 0.3 in tongue; P = 2.0e-7, Kruskal-Wallis test). Fusobacterium periodonticum, Saccharibacteria sp. 352, Streptococcus oralis subsp . dentisani, Prevotella melaninogenica, Granulicatella adiacens, Campylobacter concisus, and Haemophilus parainfluenzae were the core operational taxonomic units (OTUs) common to both sites. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differed significantly in terms of bacterial composition, they showed inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.