Journal of Clinical Medicine

Review The Role of Oral in Intra-Oral Halitosis

Katarzyna Hampelska 1,2, Marcelina Maria Jaworska 1 , Zuzanna Łucja Babalska 3 and Tomasz M. Karpi ´nski 3,*

1 Department of Genetics and Pharmaceutical , Pozna´nUniversity of Medical Sciences, Swi˛ecickiego4,´ 60-781 Pozna´n,Poland; [email protected] (K.H.); rufi[email protected] (M.M.J.) 2 Central Microbiology Laboratory, H. Swi˛ecickiClinical´ Hospital, Pozna´nUniversity of Medical Sciences, Przybyszewskiego 49, 60-355 Pozna´n,Poland 3 Chair and Department of Medical Microbiology, Pozna´nUniversity of Medical Sciences, Wieniawskiego 3, 61-712 Pozna´n,Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-61-854-6138

 Received: 27 June 2020; Accepted: 31 July 2020; Published: 2 August 2020 

Abstract: Halitosis is a common ailment concerning 15% to 60% of the human population. Halitosis can be divided into extra-oral halitosis (EOH) and intra-oral halitosis (IOH). The IOH is formed by volatile compounds, which are produced mainly by anaerobic . To these odorous substances belong volatile sulfur compounds (VSCs), aromatic compounds, amines, short-chain fatty or organic acids, alcohols, aliphatic compounds, aldehydes, and ketones. The most important VSCs are hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, and methyl mercaptan. VSCs can be toxic for human cells even at low concentrations. The oral bacteria most related to halitosis are spp., spp., Dialister spp., Eubacterium spp., spp., Leptotrichia spp., spp., Porphyromonas spp., spp., Selenomonas spp., Solobacterium spp., , and Veillonella spp. Most bacteria that cause halitosis are responsible for periodontitis, but they can also affect the development of oral and digestive tract cancers. Malodorous agents responsible for carcinogenesis are hydrogen sulfide and acetaldehyde.

Keywords: halitosis; malodor; volatile sulfur compounds; hydrogen sulfide; microbiota; Fusobacterium; Porphyromonas; Prevotella; periodontitis; carcinogenesis

1. Introduction Halitosis is a common problem that manifests as an unpleasant and disgusting odor emanating from the mouth [1]. Malodor is mainly caused by putrefactive actions of on endogenous or exogenous proteins and peptides. Oral malodor is an embarrassing condition that affects a large percentage of the human population. This condition often results in nervousness, humiliation, and social difficulties, such as the inability to approach people and speak to them [2–6]. Halitosis experiences from about 15% to 60% of the human population worldwide [7–12]. Halitosis can be divided into extra-oral halitosis (EOH) and intra-oral halitosis (IOH) [2,3,5]. The factors that increase the likelihood of halitosis include periodontal diseases, dry mouth, smoking, alcohol consumption, dietary habits, diabetes, and obesity. Halitosis can also be affected by the general hygiene of the body (i.e., dehydration, starvation, and high physical exertion), advanced age, bleeding , decreased brushing frequency, but also by stress [3,13–16]. Produced during stress, catecholamines and cortisol increased hydrogen sulfide production by sub-gingival anaerobic bacteria [17]. The medications which can cause extra-oral halitosis were categorized into 10 groups: acid reducers, aminothiols, anticholinergics, antidepressants, antifungals, antihistamines and steroids, antispasmodics, chemotherapeutic agents, dietary supplements, and organosulfur substances [18].

J. Clin. Med. 2020, 9, 2484; doi:10.3390/jcm9082484 www.mdpi.com/journal/jcm J. Clin.J. Clin. Med. Med.2020 2020, 9, ,9 2484, x FOR PEER REVIEW 2 of2 18 of 17

More and more patients are struggling with bad breath and report this problem to their primaryMore andcare more practitioner patients arefor strugglingdiagnosis withand badmanagement breath and report[19,20]. this However, problem tomany their physicians, primary care practitionerdentists, and for biologists diagnosis andhave management insufficient knowledge [19,20]. However, regarding many the physicians, cause and dentists,biochemistry and biologists of this havedisease. insu ffi cient knowledge regarding the cause and biochemistry of this disease. InIn this this review, review, we we focused focused on on intra-oral intra-oral halitosis,halitosis, regardlessregardless of of classification. classification.

2.2. Classifications Classifications of of Halitosis Halitosis InIn the the literature, literature, mainly mainly threethree classificationsclassifications of of halitosis halitosis are are used, used, described described by by Miyazaki Miyazaki et etal., al., 19991999 [21 [21],], Tangerman Tangerman andand WinkelWinkel inin 20102010 [[22],22], and Aydin Aydin and and Harvey-Woodworth Harvey-Woodworth in in 2014 2014 [23] [23 ] (Figure(Figure1). 1).

FigureFigure 1.1. ClassificationsClassifications of of halitosis halitosis [21–24]. [21–24].

MiyazakiMiyazaki et et al. al. divided divided halitosis halitosis as intra-oralas intra-oral (IOH) (IOH) and extra-oraland extra-oral (EOH) (EOH) [21]. Extra-oral [21]. Extra-oral halitosis canhalitosis be of can bloodborne be of bloodborne or non-bloodborne or non-bloodborne origin orig andin and covers covers about about 5–10% 5–10% of all all halitosis halitosis [22]. [22 ]. Bloodborne-relatedBloodborne-related causes causes include include diabetes diabetes metabolic metabo disorders,lic disorders, kidney kidney and and liver liver diseases, diseases, and certainand drugscertain and drugs food. and Non-bloodborne-related food. Non-bloodborne-related causes includecauses include respiratory respiratory and gastrointestinal and gastrointestinal diseases. Meanwhile,diseases. Meanwhile, pathological pathological conditions conditions in the oral in cavitythe oral are cavity responsible are responsible for 80–90% for 80–90% of IOH of [ 2IOH,3,25 ]. Both[2,3,25]. aerobic Both and aerobic anaerobic and bacteria anaerobic can bacteria be responsible can be forresponsible IOH. These for microorganisms IOH. These microorganisms tend to produce foul-smelling,tend to produce sulfur-containing foul-smelling, gases sulfur-containing called volatile gases sulfur called compounds volatile (VSCs)sulfur compounds [23,26]. (VSCs) [23,26].In the classification of Tangerman and Winkel [22], halitosis is classified as genuine and delusional. DelusionalIn the halitosis classification (monosymptomatic of Tangerman hypochondriasis; and Winkel [22], imaginary halitosis halitosis) is classified is a conditionas genuine in whichand patientsdelusional. believe Delusional that their halitosis breath is (monosymptomatic smelly and offensive. hypochondriasi The social pressures; imaginary of having halitosis) fresh smelling is a condition in which patients believe that their breath is smelly and offensive. The social pressure of breath increases the number of people that are preoccupied with this condition. However, the perception having fresh smelling breath increases the number of people that are preoccupied with this of oral malodor does not always reflect actual clinical oral malodor [27]. Self-perceived halitosis was condition. However, the perception of oral malodor does not always reflect actual clinical oral found to be more prevalent amongst males, particularly smokers, compared to females. However, malodor [27]. Self-perceived halitosis was found to be more prevalent amongst males, particularly there are no statistical differences when comparing with different age groups [28]. Genuine halitosis is smokers, compared to females. However, there are no statistical differences when comparing with further subdivided into physiological and pathological halitosis. Physiological halitosis (foul morning different age groups [28]. Genuine halitosis is further subdivided into physiological and breath, morning halitosis) is caused by saliva retention, as well as the putrefaction of entrapped food pathological halitosis. Physiological halitosis (foul morning breath, morning halitosis) is caused by particles.saliva retention, Meanwhile, as well intra- as and the extra-oral putrefaction causes of entrapped are responsible food forparticles. pathological Meanwhile, halitosis intra- [3,4 ,and19]. extra-oralAydin andcauses Harvey-Woodworth are responsible for dividedpathological pathologic halitosis halitosis [3,4,19]. into five types: Type 1 (oral), Type 2 (airway), Type 3 (gastroesophageal), Type 4 (blood-borne) and Type 5 (subjective). Moreover, it is Type 0 halitosis (physiologic odor), which can be a connection of the physiologic contributions of oral, J. Clin. Med. 2020, 9, 2484 3 of 17 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3 of 18 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3 of 18 J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3 of 18 airway, gastroesophageal,Aydin and Harvey-Woodworth blood-borne, divided and subjective pathologic halitosis. halitosis into Any five combination types: Type of1 (oral), the above Type types Aydin and Harvey-Woodworth divided pathologic halitosis into five types: Type 1 (oral), Type 2 (airway),Aydin Typeand Harvey-Woodworth 3 (gastroesophageal), divided Type 4 pathologic (blood-borne) hali tosisand Typeinto five 5 (subjective). types: Type Moreover, 1 (oral), Type it is can be2 (airway), present Type in every 3 (gastroesophageal), healthy person [Type23]. 4 (blood-borne) and Type 5 (subjective). Moreover, it is Type2 (airway), 0 halitosis Type (physiologic3 (gastroesophageal), odor), which Type can 4 (blood-borne) be a connection and ofType the 5physiologic (subjective). contributions Moreover, it ofis Type 0 halitosis (physiologic odor), which can be a connection of the physiologic contributions of 3. Volatileoral,Type airway, 0 Compoundshalitosis gastroesophageal, (physiologic odor), blood-borne, which can and be subjective a connection halitosis. of the Any physiologic combination contributions of the above of oral, airway,J. Clin. Med. gastroesophageal, 2020, 9, x FOR PEER REVIEW blood-borne, and subjective halitosis. Any combination of the3 of above 18 typesoral, airway, can be presentgastroesophageal, in every healthy blood-borne, person [23].and subjective halitosis. Any combination of the above typesHalitosis can be is present formed in by every volatile healthy compounds, person [23]. which are produced mainly by bacteria in the oral Aydin and Harvey-Woodworth divided pathologic halitosis into five types: Type 1 (oral), Type cavity.3. Volatile In2 the(airway), oralCompounds cavity,Type 3 (gastroesophageal), nearly 700 different Type compounds4 (blood-borne) haveand Type been 5 (subjective). detected [Moreover,29]. To these it is volatile 3. Volatile Compounds substancesHalitosisType belong 0 halitosis is sulfurformed (physiologic compounds,by volatile odor), compounds, which aromatic can bewhich compounds, a connection are produced of amines, the mainlyphysiologic short-chain by bacteriacontributions fatty in the of or oral organic Halitosisoral, airway, is formedgastroesophageal, by volatile blood-borne, compounds, and which subjective are producedhalitosis. Any mainly combination by bacteria of the in above the oral acids,cavity. alcohols,Halitosis In the aliphatic oralis formed cavity, compounds, by nearly volatile 700 compounds,different aldehydes, compounds which and are ketones have produced been (Table detected mainly1)[ 25 by[29].,30 bacteria– To33 ].these Itin is thevolatile considered oral cavity.types In the can oralbe present cavity, in nearly every healthy700 different person compounds[23]. have been detected [29]. To these volatile thatsubstancescavity. hydrogen In the belong sulfide, oral cavity, sulfur methyl nearlycompou mercaptan, 700nds, different aromatic and compounds dimethylcompounds, have sulfide amines been are detected, short-chain the main [29]. To volatilefatty these or organicvolatile compounds substances belong sulfur compounds, aromatic compounds, amines, short-chain fatty or organic in IOHacids,substances [34 alcohols,–37 ].belong In aliphatic many sulfur studies, compounds, compou thends, measurementaldehydes, aromatic compounds,and ofketones malodor amines(Table substances ,1) short-chain [25,30–33]. concerns faIttty is consideredor onlyorganic volatile acids,3. alcohols, Volatile Compounds aliphatic compounds, aldehydes, and ketones (Table 1) [25,30–33]. It is considered sulfurthatacids, compounds hydrogen alcohols, sulfide, aliphatic (VSCs). methyl compounds, The mercaptan, most aldehydes, commonly and dimethyl and used ketones sulfide are VSC(Table are the monitors, 1) main [25,30–33]. volatile such It compounds is as considered the Halimeter in that hydrogenHalitosis sulfide, is formed methyl by volatilemercaptan, compounds, and dimethyl which are sulfide produced are the mainly main by volatile bacteria compounds in the oral in (Interscan,IOH [34–37]. Chatsworth, In many USA)studies, [11 the,36 measurement,38–41]. This of method malodor has substances a significant concerns disadvantage only volatile becausesulfur the IOH [34–37].cavity. In In the many oral cavity,studies, nearly the measurement700 different compounds of malodor have substances been detected concerns [29]. onlyTo these volatile volatile sulfur measurecompounds of dimethyl (VSCs). sulfide The most is not commo exactnly [42 used]. Moreover, are VSC monitors, the presence such ofas alcohols,the Halimeter phenyl (Interscan, compounds, compoundssubstances (VSCs). belong The sulfur most compou commonds,nly aromatic used are compounds, VSC monitors, amines such, short-chain as the Halimeter fatty or organic(Interscan, Chatsworth,acids, alcohols, USA) [11,36,38–41].aliphatic compounds, This method aldehydes, has anda significant ketones (Table disadvantage 1) [25,30–33]. because It is considered the measure and polyaminesChatsworth, USA) can interfere [11,36,38–41]. with This readings method [16 has,43 ].a Forsignificant this reason, disadvantage in the assessmentbecause the ofmeasure IOH, other of dimethylthat hydrogen sulfide sulfide, is not methyl exact [42].mercaptan, Moreover, and dimethyl the presence sulfide of are alcohols, the main phenylvolatile compounds,compounds in and substancesof dimethyl are oftensulfide not is not taken exact into [42]. account. Moreover, However, the presence they of canalcohols, have phenyl an equally compounds, important and role. polyaminesIOH [34–37]. can interfereIn many studies, with readings the measurement [16,43]. ofFor malodor this reason, substances in the concerns assessment only volatile of IOH, sulfur other It is confirmedpolyamines bycan studies interfere using with gasreadings chromatography-mass [16,43]. For this reason, spectrometry in the assessment [29,32,44 of]. IOH, In the other paper of substancespolyaminescompounds are can often (VSCs).interfere not The taken with most into readings commo account.nly [16,43]. used However, are For VSC this they monitors, reason, can have suchin anthe as equally theassessment Halimeter important of (Interscan, IOH, role. other It is substances are often not taken into account. However, they can have an equally important role. It is MonedeiroconfirmedsubstancesChatsworth, et al., areby in studiesoften theUSA) personsnot [11,36,38–41].using taken withgas into chromatography-m IOH, account.This method 85 volatiles, However, has a weresignificantass they spectrometry detected,can disadvantagehave an and [29,32equally the because most,44]. important In predominantthe the measure paperrole. It ofis classes confirmed by studies using gas chromatography-mass spectrometry [29,32,44]. In the paper of of malodorMonedeiroconfirmedof dimethyl compounds byet al.,studies sulfide in the were usingis persons not alcohols exactgas with chromatography-m[42]. andIOH, Moreover, ketones. 85 volatiles, the Inpresenceass this werespectrometry group, ofdetected, alcohols, in comparison [29,32and phenyl the,44]. compounds,most In to thepredominant healthy paper and persons,of Monedeiro et al., in the persons with IOH, 85 volatiles, were detected, and the most predominant an increasedclassesMonedeiropolyamines of numbermalodor et al., can in ofcompounds interferethe volatile persons with sulfur were with readings compoundsalcohols IOH, [16,43]. 85 and volatiles, For keto and thisnes. esters were reason, In thisdetected, was in group, observed.the assessmentand in comparisonthe Simultaneously, most of IOH, predominant to other healthy authors classessubstances of malodor are oftencompounds not taken were into account.alcohols However, and keto theynes. canIn thishave group, an equally in comparison important role. to healthyIt is foundpersons,classes ten VSCsof malodoran substances:increased compounds number methyl were of thioacetate, alcohols volatile and sulfur dimethylketo nes.compounds In disulfide, this group, and dimethyl in esterscomparison trisulfide,was toobserved. healthy dimethyl persons,confirmed an increasedby studies numberusing gas ofchromatography-m volatile sulfurass compoundsspectrometry and[29,32 ,44].esters In thewas paper observed. of tetrasulfide,Simultaneously, dimethyl authors pentasulfide, found ten dimethyl VSCs sulfone,substances: allyl methyl thiocyanate, thioacetate, allyl isothiocyanate,dimethyl disulfide, S-methyl Simultaneously,Monedeiro et authorsal., in the found persons ten with VSCs IOH, 85substances: volatiles, weremethyl detected, thioacetate, and the mostdimethyl predominant disulfide, pentanethioate,dimethyl trisulfide, and thiolan-2-one dimethyl [tetrasulfide,44]. In other dimethyl studies, inpentasulfide, halitosis patients, dimethyl the sulfone, 30 most abundantallyl dimethylclasses trisulfide, of malodor compoundsdimethyl tetrasulfide,were alcohols anddimethyl ketones. pentasulfide,In this group, indimethyl comparison sulfone, to healthy allyl thiocyanate,persons, allylan isothiocyanate,increased number S-methyl of volatile pentanethioate, sulfur compounds and thiolan-2-one and esters [44]. was In otherobserved. studies, volatilethiocyanate, compounds allyl in isothiocyanate, the oral cavity S-methyl belonged pentanethioate, to alkanes or alkaneand thiolan-2-one derivatives, [44]. therein In other methyl studies, benzene, in halitosisSimultaneously, patients, authorsthe 30 foundmost abundantten VSCs volasubstances:tile compounds methyl thioacetate, in the oral dimethyl cavity disulfide,belonged to tetramethylin halitosis butane, patients, and the ethanol 30 most [45 ].abundant Dadamio vola ettile al. reportedcompounds VSC in andthe oral amines cavity (such belonged as putrescine, to alkanesdimethyl or alkane trisulfide, derivatives, dimethyl therein tetrasulfide, methyl dimethylbenzene, pentasulfide,tetramethyl dimethylbutane, andsulfone, ethanol allyl [45]. alkanes or alkane derivatives, therein methyl benzene, tetramethyl butane, and ethanol [45]. cadaverine,Dadamioalkanesthiocyanate, andor et alkaneal. trimethylamine) reported allyl derivatives, isothiocyanate, VSC and astherein theamines S-methyl most methyl (such pentanethioate, abundant asbenzene, putrescine, organic andtetramethyl thiolan-2-onecadaverine, compounds butane, and[44]. in trimethylamine)In IOHand other ethanol patients studies, [45]. [ 46as ]. Dadamio et al. reported VSC and amines (such as putrescine, cadaverine, and trimethylamine) as theDadamioIn Tablemostin halitosis abundant 1et, amongal. reportedpatients, organic others, VSCthe compounds values30 and most amines ofabundant odorin IOH(such thresholds patientsvola as tileputrescine, compounds [46]. are presented. cadaverine, in the oral Amidand cavity trimethylamine) VSCs, belonged which to areas the the most abundant organic compounds in IOH patients [46]. mostthe often mostInalkanes describedTable abundant or1, amongalkane compoundsorganic derivatives,others, compounds values in therein IOH, of in odor theIOHmethyl lowestthresholds patients benzene, value [46]. are tetramethyl of presented. odor threshold butane, Amid VSCs,and has ethanol methylwhich [45]. are mercaptan, the In Table 1, among others, values of odor thresholds are presented. Amid VSCs, which are the followedmostIn Dadamiooften by Table hydrogen described 1,et amongal. reported sulfidecompounds others, VSC and values and in dimethyl IOH,amines of odor the (such lowest sulfide. thresholds as putrescine,value This are of meansodorpresented. cadaverine, threshold that Amid theseand has trimethylamine) VSCs, substancesmethyl which mercaptan, are areas the mainly most theoften most described abundant compounds organic compounds in IOH, in the IOH lowest patients value [46]. of odor threshold has methyl mercaptan, responsiblefollowedmost often for by described thehydrogen unpleasant compounds sulfide smell and in dimethyl inIOH, the the mouth. lowestsulfide.Besides, value This ofmeans odor methyl thatthreshold mercaptanthese hassubstances methyl is felt mercaptan,are in muchmainly lower followed Inby Table hydrogen 1, among sulfide others, and values dimethyl of odor sulfid thresholdse. This are means presented. that theseAmid substancesVSCs, which are are mainlythe responsiblefollowed by for hydrogen the unpleasant sulfide smelland dimethyl in the mouth. sulfid Besides,e. This means methyl that mercaptan these substances is felt in much are mainly lower concentrationsresponsiblemost often thanfor describedthe the unpleasant other compounds compounds. smell in in IOH, the themouth. lowest Besides, value of methyl odor threshold mercaptan has methylis felt in mercaptan, much lower concentrationsresponsible for thanthe unpleasant the other compounds. smell in the mouth. Besides, methyl mercaptan is felt in much lower concentrationsfollowed by than hydrogen the other sulfide compounds. and dimethyl sulfide. This means that these substances are mainly responsible forTable the unpleasant 1. Volatile smell compounds in the mouth. present Besides, in halitosis methyl [23 mercaptan,30–33,44 is,47 felt,48 in]. much lower Table 1. Volatile compounds present in halitosis [23,30–33,44,47,48]. concentrations thanTable the 1. otherVolatile compounds. compounds present in halitosis [23,30–33,44,47,48]. Table 1. Volatile compounds present in halitosis [23,30–33,44,47,48].Odor Odor Odor Toxicity in Group of CompoundTable 1. VolatileChemical compounds present in halitosis [23,30–33,44,47,48].ThresholdOdor Toxicity in Group of Compound Chemical Chemical Structure ThresholdOdor ToxicityRats LDin 50 CompoundsGroup of CompoundName ChemicalFormula Chemical Structure Threshold(ppm) ToxicityRats LD 50in Group of Compound Chemical Threshold 50 Compounds Name Formula Chemical Structure (ppm) Rats(mg LD/50kg) Compounds Name Formula Chemical Structure [Odor49(ppm)–52 ] Rats(mg/kg) LD50 Compounds Name Formula [49–52](ppm) Toxicity(mg/kg) in Group of Compound Chemical Threshold[49–52] (mg/kg) Chemical Structure [49–52] Rats LD50 Compounds HydrogenHydrogenName Formula (ppm)[49–52] Hydrogen HH2S S 0.000040.00004 (mg/kg)15 15[53] [ 53] Hydrogen 2 2 sulfidesulfide H2S [49–52]0.00004 15 [53] sulfide H2S 0.00004 15 [53] sulfideHydrogen 61 (unspecified Methyl H2S 0.00004 611561 (unspecified[53] (unspecified MethylMethylsulfide CH4S 5.1 × 10−13 61 (unspecifiedmammal Methyl 4 −1313 mercaptan CHCH4S4 S 5.15.1 ×10 10−−13 mammalmammal mercaptanmercaptan CH4S 5.1 × 10 61 (unspecifiedspecies) [54] mercaptanMethyl × species) [54] 4 −13 species)species) [54] [ 54] CH S 5.1 × 10 mammalspecies) [54] Volatile sulfur mercaptan Volatile sulfur Dimethyl species) [54] Volatile sulfur Dimethyl C2H6S 0.00012 3300 [54,55] compounds DimethylDimethyl C2H6S 0.00012 3300 [54,55] compoundsVolatile sulfur sulfide CC2HH6S S 0.000120.00012 3300 3300 [54,55] [54, 55] compounds(VSC) sulfidesulfideDimethyl C2H2 6S6 0.00012 3300 [54,55] (VSC)compounds sulfide C2H6S 0.00012 3300 [54,55] Volatile(VSC) sulfur sulfide compoundsJ. Clin. Med.(VSC) 2020, 9, x FORDimethyl PEER REVIEW 4 of 18 Dimethyl C2H6S2 0.00029 190 [54] DimethylDimethyldisulfide C2H6S2 0.00029 190 [54] (VSC) disulfideDimethyl CC2HH6S2S 0.000290.00029 190 190 [54] [ 54] disulfide 2C2H66S22 0.00029 190 [54] disulfidedisulfidedisulfide Odor Dimethyl Toxicity in Group of CompoundDimethyl ChemicalC2H6S3 Thresholdno data no data Dimethyl 2 6 3 Chemical Structure Rats LD50 DimethyltrisulfideDimethyl C2H6S3 no data no data Compounds trisulfideName FormulaCC2HCH26HS36S S 3 nonono data(ppm) data data no data no no data data trisulfidetrisulfidetrisulfide 2 6 3 (mg/kg) [49–52]

AllylAllyl methyl methyl CC4HH8S S 0.000140.00014 no nodata data sulfidesulfide 4 8

Pyridine C5H5N 0.01 360–891 [54,55]

Picoline C6H7N 0.0026 200–790 [54,55]

Aromatic compounds Indole C8H7N 0.0003 1000 [54,55]

Skatole C9H9N 0.0000056 3450 [54,55]

Ammonia H3N 0.043 350 [56]

567–8471 Urea CH4N2O no data [54,55]

Methylamine CH5N 0.00075 100 [54,55]

Dimethylamine C2H7N 0.00076 698 [54,55,57]

Amines

Trimethylamine C3H9N 0.00002 500–535 [54,55]

463–2000 Putrescine C4H12N no data [54,55,58]

Cadaverine C5H14N no data 2000 [58]

Short/medium fatty or organic Acetic acid C2H4O2 0.0004 3310 [54,55] acids

J. Clin. Med. 2020, 9, x FOR PEER REVIEW 4 of 18

Odor Toxicity in Group of Compound Chemical Threshold J. Clin. Med. 2020, 9, x FOR PEER REVIEW Chemical Structure Rats4 of LD 18 50 CompoundsJ. Clin. Med. 2020, 9, xName FOR PEER REVIEWFormula (ppm) 4 of 18 J. Clin. Med. 2020, 9, x FOR PEER REVIEW (mg/kg)4 of 18 J. Clin. Med. 2020, 9, x FOR PEER REVIEW [49–52] 4 of 18 Odor[49–52] Odor Toxicity in Group of Compound Chemical ThresholdOdor Toxicity in J. Clin. Med. 2020Group, 9, 2484of Compound Chemical Chemical Structure Threshold Rats LD50 4 of 17 Chemical Structure Odor ToxicityRats LD in50 CompoundsGroup of AllylCompound methylName ChemicalFormula Threshold(ppm) Toxicity in Compounds Allyl methylName Formula Chemical Structure (ppm) Rats(mg/kg) LD50 GroupJ.J. Clin.Clin.of Med.Med. 20202020Compound,, 99,, xx FORFOR PEERPEER C REVIEWREVIEW4HChemical8S [49–52]Threshold0.00014 44 ofof (mg/kg) 1818 no data CompoundsJ. Clin. Med.sulfide 2020Name, 9 , x FOR PEER REVIEWFormula Chemical Structure [49–52](ppm) 4 of 18Rats LD50 Compounds sulfide Name Formula (ppm) (mg/kg) [49–52] (mg/kg) OdorOdor [49–52] J. Clin. Med. 2020, 9, x FOR PEER REVIEW Odor ToxicityToxicity inin4 of 18 GroupGroup ofofAllyl methylCompoundCompound ChemicalChemicalTable 1. Cont. ThresholdThreshold Toxicity in Group ofAllyl methylCompound C4HChemical8S ChemicalChemical StructureStructure Threshold0.00014 RatsRats LDLD5050 no data CompoundsCompounds NameName C4HFormula8FormulaS Chemical Structure (ppm)(ppm)0.00014 Rats LD50 no data CompoundsAllyl sulfide methyl Name Formula (ppm) (mg/kg)(mg/kg) sulfide C4H8S [49–52]Odor0.00014 (mg/kg) no data Allyl methyl [49–52][49–52] Odor Toxicity in Group of sulfideCompound ChemicalC4H8S Threshold0.00014 no data sulfide Chemical Structure Rats LD50 Toxicity in Group of CompoundsCompoundPyridine Name ChemicalC5H5NFormula (ppm)Threshold 0.01 360–891 [54,55] J. Clin. Med. 2020, 9, x FORAllylAllyl PEER methylmethyl REVIEW Chemical Structure (mg/kg)4 of 18 Rats LD50 Compounds Name Allyl methylFormula CC44HH88SS 0.000140.00014[49–52] (ppm) nono datadata sulfide C4H8S 0.00014 no data sulfidesulfide (mg/kg) Odor[ 49–52] Pyridine C5H5N 0.01 Toxicity360–891 in [54,55] Group of PyridineAllylCompound methyl C5HChemical5N Threshold0.01 360–891 [54,55] Chemical Structure Rats LD50 CompoundsPyridine Name C5HC5FormulaN4H 8S (ppm)0.000140.01 no360–891 data [54,55] Pyridinesulfide C5H5N 0.01 (mg/kg)360–891 [54,55] [49–52] PyridinePyridinePyridine C 5H5NCC55HH55NN 0.010.01 0.01360–891360–891 [54,55][54,55] 360–891 [54,55] Picoline PyridineC 6H7N C5H5N 0.01 0.0026360–891 [54,55]200–790 [54,55]

Allyl methyl C4H8S 0.00014 no data Picoline sulfide C6H7N 0.0026 200–790 [54,55] PicolinePyridine C6HC75NH 5N 0.010.0026 360–891200–790 [54,55] [54,55] Picoline C6H7N 0.0026 200–790 [54,55] Picoline C6H7N 0.0026 200–790 [54,55] PicolinePicolinePicoline C H NCC66HH77NN 0.00260.0026 0.0026200–790200–790 [54,55][54,55] 200–790 [54,55] Picoline 6 7 C6H7N 0.0026 200–790 [54,55] Aromatic

compounds Pyridine C5H5N 0.01 360–891 [54,55] Aromatic AromaticAromatic Indole C8H7N 0.0003 1000 [54,55] Indole Picoline C H NC 6H7N 0.00260.0003 200–790 [54,55]1000 [54,55] compoundsAromatic AromaticAromatic compoundscompoundsAromatic Aromatic compoundscompounds Indole C8H7N 0.0003 1000 [54,55] compounds compounds Indole C8H7N 0.0003 1000 [54,55] compounds IndoleIndole CC88HH77NN 0.00030.0003 10001000 [54,55][54,55] IndoleIndole Indole C8CH8H77NNC8 H7N 0.00030.0003 0.0003 1000 [54,55]1000 [54,55] 1000 [54,55] Indole C8H7N 0.0003 1000 [54,55] Picoline C6H7N 0.0026 200–790 [54,55] Aromatic

compounds

Indole C8H7N 0.0003 1000 [54,55] Aromatic compoundsSkatole C9H9N 0.0000056 3450 [54,55] Skatole SkatoleSkatole C 9H9N CC99HH99NN 0.00000560.00000560.0000056 34503450 [54,55] [54,55] 3450 [54,55] SkatoleIndole C89H79N 0.00000560.0003 34501000 [54,55] [54,55] SkatoleSkatole C9CH9H99NN 0.00000560.0000056 3450 [54,55] 3450 [54,55] Skatole C9H9N 0.0000056 3450 [54,55] Skatole C9H9N 0.0000056 3450 [54,55] Skatole C9H9N 0.0000056 3450 [54,55]

Ammonia H3N 0.043 350 [56] SkatoleAmmonia C9HH93NN 0.0430.0000056 3503450 [56] [54,55] Ammonia H3N 0.043 350 [56] AmmoniaAmmonia H H3NN 0.0430.043 350 350 [56] [ 56] AmmoniaAmmonia H3NH3 3 N 0.0430.043 350 [56]350 [56] Ammonia H3N 0.043 350 [56] 567–8471 3 567–8471 AmmoniaSkatole UreaUrea H NCHCHC 39H44NN9N22OO 0.0000056nono datadata0.043 3450567–8471 [54,55] 350 [56] AmmoniaUrea HCHN4N 2O no data 0.043 [54,55] 350 [56] [54,55][54,55] 567–8471567–8471567–8471 Ammonia H3N 0.043 350 [56] 567–8471 UreaUreaUrea CH CHCH4NN42NO2 O nonono data data data 567–8471 Urea CH4 4N22O no data 567–8471[54,55][54,55] 5 567–8471[54,55][54 , 55] UreaMethylamineMethylamine CH 4N2CHCHO 55NN 0.000750.00075no data 100100 [54,55][54,55] [54,55] UreaMethylamine CHCH4N25ON 0.00075 no data100 [54,55] [54,55][54,55] Ammonia H3N 0.043 350567–8471 [56] Urea CH4N2O no data DimethylamineDimethylamine CC22HH77NN 0.000760.00076 698698 [54,55,57][54,55,57][54,55] MethylamineDimethylamine CH 5NC 2H7N 0.000760.00075 698 [54,55,57]100 [54,55] MethylamineMethylamine CHCH5N5 N 0.000750.00075 100 100[54,55] [54,55] MethylamineAminesAminesMethylamine CH CH5N5N 0.000750.00075567–8471 100 100 [54,55] [54, 55] Amines Urea 5CH4N2O no data MethylamineMethylamine CHCHN 5N 0.000750.00075 [54,55]100 100 [54,55] [54,55]

Methylamine CH5N 0.00075 100 [54,55] DimethylamineTrimethylamineTrimethylamine C 2H7NCC33 HH99NN 0.000020.000020.00076 500–535500–535 [54,55][54,55]698 [54,55,57] DimethylamineTrimethylamine C 2H7NC3 H9N 0.000020.00076 500–535 [54,55]698 [54,55,57] Amines Dimethylamine C2H7N 0.00076 698 [54,55,57] DimethylamineDimethylamine C2CH2H77NN 0.000760.00076 698 [54,55,57] 698 [54 ,55,57] Dimethylamine C2H7N5 0.00076 698 [54,55,57] Amines Methylamine CH N 0.00075 100 [54,55] Amines Dimethylamine C2H7N 0.00076 698 [54,55,57] AminesAmines Amines Amines TrimethylamineDimethylamine C 3H9NC2 H7N 0.000760.00002 698463–2000463–2000 [54,55,57]500–535 [54,55] TrimethylaminePutrescinePutrescine C3H9CNC44H H1212NN nono datadata0.00002 463–2000500–535 [54,55] Putrescine 3 9 C4H12N no data [54,55,58][54,55,58] TrimethylamineTrimethylamineAminesTrimethylamine Trimethylamine CC 3HCH39HNC9 N3NH 9N 0.000020.000020.00002 [54,55,58] 500–535500–535 500–535 500–535 [54,55] [54,55] [54,55] [ 54, 55] Trimethylamine 3 C9 3H9N 0.00002 500–535 [54,55]

Trimethylamine C3H9N 0.00002 500–535 [54,55] CadaverineCadaverine CC55HH1414NN nono datadata 2000 2000 [58][58] Cadaverine C5H14N no data 2000 [58]

463–2000 Short/mediumShort/mediumPutrescine C4H12N no data Short/mediumPutrescine C4H12N no data 463–2000[54,55,58]463–2000463–2000 463–2000 fatty or organic Acetic acid C2H4O2 0.0004 3310 [54,55][54,55,58] fattyfatty oror organicorganicPutrescine PutrescinePutrescineAceticAcetic acidacid C4HC124 CH CN2H1212H N4OO 2 0.00040.0004no nodata no data data3310 3310 [54,55][54,55] 463–2000 fattyPutrescine orPutrescine organic Acetic acidC C4 H4H1212N CN2H4O2 0.0004no no data data3310 [54,55,58] [54,55] acidsacidsPutrescine C4H12N no data463–2000 [54,55,58] [54,55,58][54 ,55 ,58] acids Putrescine C4H12N no data [54,55,58] [54,55,58]

Cadaverine C5H14N no data 2000 [58] Cadaverine C5H14N no data 2000 [58] Cadaverine C5H14N no data 2000 [58] CadaverineCadaverineCadaverine C CH5HC145HN14 N nonono data data data 2000 2000 [58] 2000[58] [58] Cadaverine 5 14C5H14N no data 2000 [58] J. Clin. CadaverineMed. 2020, 9, x FOR PEERC REVIEW5H14N no data 5 of 18 2000 [58] Short/mediumJ. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18 Short/mediumShort/mediumJ. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18 fattyShort/mediumShort/medium or organic Acetic acid C2H4O2 0.0004 3310 [54,55] fatty or fattyorganic Short/mediumor organic Acetic Acetic acid acid C2H C42OH24 O2 Odor0.00040.0004 3310 3310[54,55] [54,55] Odor Toxicity in Short/mediumfattyfatty acidsor organic or organicfattyacids or organic Acetic Acetic acidAcetic acid acid C2H C42O CH22H4 O4O2 2 0.0004Odor0.0004 0.0004 3310 [54,55]33103310 [54,55] [54,55] Short/mediumacids GroupAcetic of acidCompound C 2H4ChemicalO2 ThresholdOdor 0.0004 Toxicity in 3310 [54,55] Group of Compound Chemical Chemical Structure Threshold ToxicityRats LD in50 fatty or organicacidsacids Compounds GroupacidsAcetic of acid CompoundName C2 H4OChemical2Formula Chemical Structure Threshold(ppm) 0.0004 ToxicityRats LD 50in 3310 [54,55] fatty or organic CompoundsGroupAcetic of acid CompoundName C2 H4OChemical2Formula Chemical Structure Threshold(ppm) 0.0004 Rats(mg/kg) LD50 3310 [54,55] Compounds Name Formula Chemical Structure (ppm) Rats LD50 acids Compounds Name Formula [49–52](ppm) (mg/kg) acids [49–52] (mg/kg) [49–52] 2600–3500 2600–3500 Propionic acid C3H6O2 0.00099 2600–3500 Propionic acidPropionic acid C3 H6O C32H6O2 0.000990.00099 2600–3500[54,55] Propionic acid C3H6O2 0.00099 2600–3500[54,55] [54,55] Propionic acid C3H6O2 0.00099 [54,55] [54,55]

Short/medium 1500–2000 Butyric acid C4H8O2 0.001 1500–2000 1500–2000 fatty or organic Butyric acid C4H8O2 0.001 1500–2000[54,55] Butyric acidButyric acid C 4H8O C42H8O2 0.001 0.001 1500–2000[54,55] Butyric acid C4H8O2 0.001 [54,55] [54,55] acids [54,55]

Valeric acid C5H10O 0.000037 2000–4600 [59] Valeric acid C5H10O 0.000037 2000–4600 [59] Valeric acidValeric acid C H CO5H10O 0.0000370.000037 2000–4600 [59] 2000–4600 [59] Valeric acid 5 10 C5H10O 0.000037 2000–4600 [59]

Isovaleric acid C5H10O 0.000078 2 [54] Isovaleric acid C5H10O 0.000078 2 [54] Isovaleric acid C5H10O 0.000078 2 [54] Isovaleric acid C5H10O 0.000078 2 [54] Isovaleric acid C5H10O 0.000078 2 [54]

2131–7529 Methanol CH4O 3.05 2131–7529 Methanol CH4O 3.05 2131–7529[54,55] Methanol CH4O 3.05 2131–7529[54,55] Methanol CH4O 3.05 [54,55] 1440–7060[54,55] Ethanol C2H6O 0.09 1440–7060 Alcohols Ethanol C2H6O 0.09 1440–7060[54,55] 2 6 1440–7060 Alcohols Ethanol C H O 0.09 [54,55] Alcohols Ethanol C2H6O 0.09 [54,55] 590–2200[54,55] Propanol C3H8O 0.031 590–2200 Propanol C3H8O 0.031 590–2200[54,55] Propanol C3H8O 0.031 590–2200[54,55] Propanol C3H8O 0.031 [54,55] [54,55] Cyclopropane C3H6 no data no data Cyclopropane C3H6 no data no data Cyclopropane C3H6 no data no data Cyclopropane C3H6 no data no data

Aliphatic Aliphatic compoundsAliphatic Cyclobutane C4H8 no data no data compoundsAliphatic Cyclobutane C4H8 no data no data compounds Cyclobutane C4H8 no data no data compounds Cyclobutane C4H8 no data no data

400– >2000 Pentane C5H12 1.29 400– >2000 Pentane C5H12 1.29 400–[54,55] >2000 Pentane C5H12 1.29 400–[54,55] >2000 Pentane C5H12 1.29 [54,55] 640–1930[54,55] Acetaldehyde C2H4O 0.0015 640–1930 Acetaldehyde C2H4O 0.0015 640–1930[54,55] Acetaldehyde C2H4O 0.0015 640–1930[54,55] Acetaldehyde C2H4O 0.0015 [54,55] [54,55] 5500–5800 Acetone C3H6O 0.4 5500–5800 Acetone C3H6O 0.4 5500–5800[54,55,57] Acetone C3H6O 0.4 5500–5800[54,55,57] Acetone C3H6O 0.4 [54,55,57] [54,55,57]

Aldehydes and 815–2650 Aldehydes and Acetophenone C8H8O 0.00024 815–2650 Aldehydes and Acetophenone C8H8O 0.00024 815–2650[54,55] Aldehydesketones and Acetophenone C8H8O 0.00024 815–2650[54,55] ketones Acetophenone C8H8O 0.00024 [54,55] ketones [54,55]

Benzophenone C13H10O no data >10,000 [54,55] Benzophenone C13H10O no data >10,000 [54,55] Benzophenone C13H10O no data >10,000 [54,55] Benzophenone C13H10O no data >10,000 [54,55]

In the oral cavity, the most relevant anatomical part related to IOH is the tongue. The tongue- In the oral cavity, the most relevant anatomical part related to IOH is the tongue. The tongue- associatedIn the microbiotaoral cavity, producethe most malodorousrelevant anatomical compounds part relatedand fatty to IOHacids. is Thethe tongue.VSCs are The the tongue- most associated microbiota produce malodorous compounds and fatty acids. The VSCs are the most essentialassociated substances microbiota responsible produce malodorous for malodor. compounds They are productsand fatty ofacids. meta Thebolism VSCs of sulfurare the amino most essential substances responsible for malodor. They are products of metabolism of sulfur amino essential substances responsible for malodor. They are products of metabolism of sulfur amino

J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18 J.J. Clin.Clin. Med.Med. 2020,, 9,, xx FORFOR PEERPEER REVIEWREVIEW 5 of 18 J.J. Clin.Clin. Med.Med. 20202020,, 99,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1818 Odor Odor Toxicity in Group of Compound Chemical ThresholdOdor Toxicity in Group of Compound Chemical Chemical Structure Threshold ToxicityToxicityRats LD in50in Group of Compound Chemical Chemical Structure Threshold Rats LD5050 CompoundsGroup of CompoundName ChemicalFormula Chemical Structure Threshold(ppm) Rats LD50 Compounds Name Formula Chemical Structure (ppm)(ppm) Rats(mg/kg) LD5050 Compounds Name FormulaFormula [49–52](ppm)(ppm) (mg/kg)(mg/kg) [49–52][49–52] (mg/kg)(mg/kg) [49–52][49–52] 2600–3500 Propionic acid C3H6O2 0.00099 2600–3500 Propionic acid C33H66O22 0.00099 2600–35002600–3500 Propionic acid C3H6O2 0.00099 2600–3500[54,55] PropionicPropionic acidacid C C33H66O22 0.000990.00099 [54,55][54,55] [54,55][54,55] J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18

Odor Toxicity in1500–2000 Group ofButyric acidCompound C4H8ChemicalO2 Threshold0.001 1500–2000 Butyric acid C44H88O22 Chemical Structure 0.001 Rats LD501500–2000 1500–2000[54,55] CompoundsButyric acidName C44H88FormulaO22 (ppm) 0.0010.001 [54,55][54,55] Butyric acid C4H8O2 0.001 (mg/kg) [54,55] [49–52] [54,55][54,55] J. Clin. Med. 2020, 9, 2484 5 of 17

J. Clin. Med. 2020, 9, x FOR PEER REVIEW 5 of 18 2600–3500 Propionic acid C3H6O2 0.00099 Valeric acid C5H10O Odor 0.000037 [54,55]2000–4600 [59] Valeric acid C55H1010O 0.000037Toxicity in 2000–4600 [59] ValericGroup of acid Compound C55H 1010O Chemical Threshold 0.000037 2000–4600 [59] Valeric acid C5H10O Table 1. Cont.Chemical Structure 0.0000370.000037Rats LD 50 2000–46002000–4600 [59][59] Compounds Name Formula (ppm) (mg/kg) [49–52] 1500–2000 Butyric acid C4H8O2 0.001Odor 2600–3500[54,55] Toxicity in Group of Compound PropionicChemical acid C3H6O2 0.00099Threshold Isovaleric acid C5H10O Chemical Structure 0.000078[54,55] 2Rats [54] LD50 Compounds IsovalericIsovalericName acidacid C CFormula55H1010O (ppm)0.000078 2 [54] Isovaleric acid C5H10O 0.000078 2 [54] IsovalericIsovaleric acidacid C C55H1010O 0.0000780.000078 22 (mg [54][54] / kg) [49–52] Valeric acid C5H10O 0.000037 1500–20002000–4600 [59] Butyric acid C4H8O2 0.001 [54,55] 2131–75292131–7529 MethanolMethanol CH CH4O4O 3.053.05 2131–7529 Methanol CH44O 3.05 2131–75292131–7529[54,55][54, 55 ] Methanol CH44O 3.053.05 [54,55][54,55] Methanol CH4O 3.05 [54,55][54,55] 1440–7060[54,55] Ethanol C2H6O 0.09 1440–70601440–7060 IsovalericValeric acid acid C5H10 CO5H 10O 0.0000370.000078 2000–46002 [59] [54] 1440–7060 AlcoholsAlcohols EthanolEthanol C C22H2H66O6 O 0.090.09 1440–70601440–7060[54,55] Alcohols Ethanol C22H66O 0.090.09 [54,55][54,55][54, 55 ] Alcohols Ethanol C2H6O 0.09 [54,55] Alcohols [54,55][54,55] 590–2200 Propanol C3H8O 0.031 2131–7529 590–2200590–2200 PropanolMethanol C33H88OCH 4O 3.05 0.031 590–2200590–2200 PropanolPropanol C C3H3H8O8 O 0.0310.031 590–2200[54,55] Propanol IsovalericC acid3H 8O C5H10O 0.000078 0.0310.0312 [54][54,55] [54,55][54,55] Propanol C3H8O 0.031 [54,55][54,55][54, 55 ] 1440–7060 [54,55] Ethanol C2H6O 0.09 Alcohols [54,55] Cyclopropane C3H6 no data2131–7529 no data Cyclopropane MethanolC33 H66 CH4O 3.05 no data no data CyclopropaneCyclopropane C C3H3H6 6 nono data data[54,55] no nodata data Cyclopropane C33H66 nono datadata 590–2200 no no datadata CyclopropanePropanol C H C 3H8O 0.031 no data no data 1440–7060[54,55] Ethanol C2H6O 0.09 Aliphatic Alcohols [54,55] Aliphatic AliphaticAliphatic Cyclobutane C4H8 no data no data compoundsAliphatic 4 8 590–2200 compounds CyclobutaneCyclopropane Propanol C4 H8 C3HC6 3H8O 0.031no data no data no data no data compounds CyclobutaneCyclobutane C C4H4H8 8 nono data data no nodata data compoundscompoundscompounds Cyclobutane C44H88 nono datadata[54,55] no no datadata compounds

Aliphatic 400– >2000 Cyclopropane5 12 C3H6 no data no data 400– >2000 PentaneCyclobutane C H C 4H8 no data 1.29 no data 400– >2000 compoundsPentane C55H1212 1.29 400–400–[54,55]400– >2000>2000> 2000 Pentane C55H1212 1.29 PentanePentanePentane CC C5H5H12 12 1.291.291.29 [54,55][54,55] Aliphatic [54,55][54,55][54, 55 ] compounds Cyclobutane C4H8 no data no data400– >2000 640–1930 Pentane 2 4 C5H12 1.29 640–1930 Acetaldehyde C H O 0.0015 640–1930 Acetaldehyde C22H44O 0.0015 [54,55] 640–1930640–1930[54,55]640–1930 Acetaldehyde C22H44O 0.00150.0015 [54,55][54,55] AcetaldehydeAcetaldehyde C C2H2H4O4 O 0.00150.0015400– >2000 [54,55][54,55] Pentane C5H12 1.29 640–1930 [54,55][54, 55] Acetaldehyde C2H4O 0.0015 [54,55] [54,55] 640–1930 Acetaldehyde C2H4O 0.0015 5500–5800 Acetone C3H6O 0.4 [54,55] 5500–5800 Acetone C33H66O 0.4 5500–58005500–5800 Acetone C3H6O 0.4 5500–5800[54,55,57]5500–5800 AcetoneAcetone C C33HH66O O 0.40.40.4 5500–5800 [54,55,57][54,55,57] Acetone 3 C63H6O 0.4 [54,55,57][54,55,57] 5500–5800[54,55,57] [54,55,57] Acetone C3H6O 0.4 [54,55,57]

Aldehydes and ketones 815–2650 Aldehydes andAldehydes Acetophenone and C8H8O 0.00024 815–2650 815–2650815–2650 Aldehydes and Acetophenone 8 8 C8H8O 0.00024 815–2650 815–2650 Aldehydes and AcetophenoneAcetophenoneAldehydes and C C8HH8O O 8 8 0.000240.00024 815–2650815–2650 Aldehydes and Acetophenone AcetophenoneC8H8 8 O8 C H O 0.00024 0.00024 [54,55] 815–2650[54,55] Aldehydesketones and ketonesAcetophenone C88H88O 0.000240.00024[54,55] [54,55][54,55][54, 55 ] ketones Acetophenoneketones C H O 0.00024 [54,55][54,55] ketonesketones [54,55]

Benzophenone C13H10O no data >10,000 [54,55] BenzophenoneBenzophenone C 13HC1013HO10O no datano data >10,000 [54,55]>10,000 [54,55]

Benzophenone C13H10O no data >10,000 [54,55] Benzophenone C1313H1010O no data >10,000 [54,55] Benzophenone C13H10O no data >10,000 [54,55] BenzophenoneBenzophenone CC1313H1010O nono datadata >10,000 >10,000 [54,55][54,55]

In the oral cavity, the most relevant anatomical part related to IOH is the tongue. The tongue- In theassociated oral cavity, microbiota the mostproduce relevant malodorous anatomical compounds part related and fatty to acids.IOH is The the VSCs tongue. are theThe most tongue- In the oral cavity, the most relevant anatomical part related to IOH is the tongue. associatedessential microbiota substances produce responsible malodorous for malodor. compounds They are andproducts fatty of acids. meta bolism The VSCsof sulfur are amino the most The tongue-associated microbiota produce malodorous compounds and fatty acids. The VSCs In theessential oral cavity, substances the most responsible relevant for malodor.anatomical They part are productsrelated toof metaIOHbolism is the of tongue. sulfur amino The tongue- are the mostIn the essentialoral cavity, substances the most relevant responsible anatomical for part malodor. related to They IOH is are the products tongue. The of tongue- metabolism associatedInIn thethe microbiotaoraloral cavity,cavity, producethethe mostmost malodorousrelevantrelevant anatomicalanatomical compounds partpart relatedrelatedand fatty toto IOHIOHacids. isis Thethethe tongue.tongue.VSCs are TheThe the tongue-tongue- most associated microbiota produce malodorous compounds and fatty acids. The VSCs are the most of sulfuressentialassociatedassociated amino substances microbiotamicrobiota acids: responsible methionine, produceproduce malodorousmalodorous for cysteine,malodor. compoundscompounds andThey homocysteineare productsandand fattyfatty of acids.acids. in meta the TheThebolism Gram-negative VSCsVSCs of sulfurareare thethe amino most anaerobicmost essential substances responsible for malodor. They are products of metabolism of sulfur amino bacteriaessentialessential [25,30 substancessubstances,47,60]. Hydrogen responsibleresponsible sulfide forfor malodor.malodor. and mercaptans ThTheyey areare are productsproducts the principal ofof metameta endbolismbolism products ofof sulfursulfur [38 ].aminoamino In healthy

volunteers, the concentration of H2S in saliva was within a range of 1.641–7.124 µM[61]. In other studies, the mean amount of H2S in the saliva of healthy persons was 0.5 ng/10 mL, whereas in patients with IOH it was 6.7 ng/10 mL [62]. Gram-positive bacteria can support Gram-negative anaerobic bacteria in the production of VSC. They cut off sugar chains from glycoproteins and provide proteins that are necessary for proteolytic processes [60]. salivarius has an impact on the deglycosylation of salivary glycoproteins, mainly mucins, which can next be degraded to VSC by [63]. In turn, Solobacterium moorei is associated with the production of VSC through β-galactosidase activity and the degradation of glycoproteins [60,64]. The essential VSCs are hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, and methyl mercaptan [25,30] (Table1). These are produced mostly by anaerobic bacteria. The increased production of malodorous gases occurs mainly in tongue coating, and diseases such as and periodontitis and, to a less extent, in pericoronitis, oral ulcers, periodontal abscesses, and herpetic gingivitis [65]. Other volatile organoleptic compounds, such as indole, skatole, amines, and ammonia, J. Clin. Med. 2020, 9, 2484 6 of 17 are produced by the putrefaction of non-sulfur containing amino acids (i.e., tryptophan, lysine and ornithine). Studies have shown that volatile sulfur compounds are the major contributors to bad breath. Hydrogen sulfide, methyl mercaptan and, to a lesser extent, dimethyl sulfide, represent 90% of the volatile sulfur compounds in halitosis [2,27]. Volatile sulfur compounds can be toxic for human cells even at low concentrations. They contain thiols (-SH groups) that interact with other proteins and support the negative interaction of bacterial antigens and enzymes. The result of this effect is chronic inflammation, periodontal gingivitis, and periodontitis [66]. In human gingival fibroblasts, H2S activates the mitochondrial pathway of apoptosis [67]. The H2S is a known genotoxic agent, which has an impact on genomic instability and cumulative mutations [68]. In studies on rats, it was demonstrated that hydrogen sulfide leads to ultrastructural changes in epithelial cells and periodontal destruction [69]. Increased amounts of H2S by the activation of proliferation, migration, and invasion can also lead to carcinogenesis [70,71]. and Porphyromonas gingivalis belong to the most essential carcinogenic oral bacteria producing VSCs [70,72]. Cancerogenic is also acetaldehyde produced from ethanol by mucosal epithelial cells or oral microflora, e.g., , Candida non-albicans, sp., and Streptococcus sp. Acetaldehyde binds to DNA and leads to the formation of DNA adducts, point mutations, and DNA cross-linking [73,74]. Other important substances causing IOH are diamines, such as putrescine and cadaverine. Both compounds are produced from amino acids, putrescine from arginine, and cadaverine from L-lysine [75,76] (Figure2). Both diamines are associated with the putrefaction of food by bacteria occurring in the and severe periodontitis [77]. Gram-negative bacteria, mostly , which can colonize the oral cavity and dentures, produce urease that hydrolyzes urea into carbon dioxide and ammonia [78]. can form ammonia from cysteine using cysteine desulfhydrase [79] or reduce nitrates to ammonia [73]. Major contributors to trimethylamine production are gut bacteria, which can be inhabitants of the oral cavity, such genera as Anaerococcus, , Collinsella, Desulfovibrio, , E. coli, Citrobacter, Edwardsiella, Providencia, and Proteus [74,80–84]. Indole and skatole are produced in high amounts by intra-oral, Gram-positive Streptococcus milleri, and anaerobic Gram-negative bacteria such as Porphyromonas intermedia, Fusobacterium nucleatum, and Porphyromonas gingivalis. Small amounts of both aromatic compounds produced Aggregatibacter aphrophilus, Staphylococcus epidermidis, and Streptococcus sanguis [85].

4. Microbiota Responsible for Intra-Oral Halitosis The human oral cavity microbiota is an ecosystem consisting of various symbiotic microbes. There is a relationship between the global composition of indigenous bacterial populations and human health [86,87]. The oral microbiota is truly diverse and consists of 50–100 billion bacteria. There are about 700 taxa, of which one-third cannot be grown in vitro [88,89]. A vast range of microorganisms inhabit the human oral cavity, including bacteria, fungi, viruses, and protozoa [90,91]. The basic oral microbiota consists of phyla, such as , , , , and . The most dominant genera are Streptococcus, Veillonella, Gemella, Granulicatella, Neisseria, , Selenomonas, Fusobacterium, Leptotrichia, Prevotella, Porphyromonas, and Lachnoanaerobaculum. Lots of current findings reported that oral bacteria can be biomarkers that differentiate healthy and pathological conditions within the oral cavity. The oral microbiota research is used as a diagnostic and prognostic tool in the aspect of human health. In the human body, the oral cavity is the second site, after the colon, containing the largest diversity of microbial populations [92]. Simultaneously, changes in the are reflected in the oral microbiota, and the microbial communities of the oral cavity and are predictive of each other [93–95]. J. Clin. Med. 2020, 9, x FOR PEER REVIEW 7 of 18 microorganisms inhabit the human oral cavity, including bacteria, fungi, viruses, and protozoa [90,91]. The basic oral microbiota consists of phyla, such as Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, and Actinobacteria. The most dominant genera are Streptococcus, Veillonella, Gemella, Granulicatella, Neisseria, Haemophilus, Selenomonas, Fusobacterium, Leptotrichia, Prevotella, Porphyromonas, and Lachnoanaerobaculum. Lots of current findings reported that oral bacteria can be biomarkers that differentiate healthy and pathological conditions within the oral cavity. The oral microbiota research is used as a diagnostic and prognostic tool in the aspect of human health. In the human body, the oral cavity is the second site, after the colon, containing the largest diversity of microbial populations [92]. Simultaneously, changes in the gut microbiota are reflected in the oral microbiota, and the microbial communities of the oral cavity and gastrointestinal tract are predictive of each other [93–95]. The oral bacteria that are most likely to produce hydrogen sulfide from L-cysteine or serum are Bacteroides spp., Eubacterium spp., Fusobacterium spp., Peptostreptococcus spp., Porphyromonas spp., Selenomonas spp., Tannerella forsythia, and Veillonella spp. Another essential component of VSC is methyl mercaptan produced from L-methionine or serum. It is a metabolic product mainly derived

J.from Clin. Med.Bacteroides2020, 9, 2484 spp., Eubacterium spp., Fusobacterium spp., Porphyromonas spp., and Treponema7 of 17 denticola [30,96] (Table 2).

Figure 2. Simplified ways of bacterial production of selected odorous compounds [30,75,76,96–102]. Figure 2. Simplified ways of bacterial production of selected odorous compounds [30,75,76,96–102].

The oral bacteria that are most likely to produce hydrogen sulfide from L-cysteine or serum are Bacteroides spp., Eubacterium spp., Fusobacterium spp., Peptostreptococcus spp., Porphyromonas spp., Selenomonas spp., Tannerella forsythia, and Veillonella spp. Another essential component of VSC is methyl mercaptan produced from L-methionine or serum. It is a metabolic product mainly derived from Bacteroides spp., Eubacterium spp., Fusobacterium spp., Porphyromonas spp., and denticola [30,96] (Table2). Ye at al.’s studies showed a correlation between high H2S and CH4S levels and the growth of microorganisms such as Prevotella spp., Peptostreptococcus spp., Eubacterium nodatum, and Alloprevotella spp. Comparing the study and control group, the authors noted significantly higher concentrations of all compounds (total VSC, H2S, CH4S, and C2H6S) in the malodor group [103]. The most active producers of hydrogen sulfide are Gram-negative anaerobes Prophyromonas gingivalis, , and Tannerella forsythia (). Furthermore, the red complex microorganisms are associated with . Hydrogen sulfide and methyl mercaptan are produced in large quantities in periodontal inflammations [104–106]. During periodontitis, Porphyromonas spp., Prevotella spp., and Treponema denticola may play the most crucial role in providing amino acids to other anaerobic bacteria. Through this process, anaerobes acquire the opportunity to produce H2S and CH4S[60] (Figure2). J. Clin. Med. 2020, 9, 2484 8 of 17

In the studies of Takeshita et al., the producers of hydrogen sulfide in saliva were bacteria from the genera Neisseria, Fusobacterium, Porphyromonas, and SR1. In contrast, producers of the methyl mercaptan are representatives of the genera Prevotella, Veillonella, Atopobium, Megasphaera, and Selenomonas [107]. Significant contributors to methyl mercaptan production are also gut bacteria, which can be inhabitants of the oral cavity, such as E. coli, Citrobacter spp., and Proteus spp. [48,84].

Table 2. Bacterial producers of volatile sulfur compounds (VSC) [30,96].

Chemical Compound Bacteria Bacteroides intermedius, Bacteroides spp., ochracea, Centipeda periodontii, , Eubacterium brachy, E. limosum, Eubacterium spp., Fusobacterium alocis, F. nucleatum, Hydrogen sulfide from L-cysteine F. periodonticum, F. sulei, Peptostreptococcus anaerobius, P. micros, P. prevotii, Porphyromonas endodontalis, Propionibacterium propionicum, Selenomonas artemidis, S. dianae, S. flueggei, S. infelix, S. noxia, S. sputigena, Tannerella forsythia, Veillonella dispar, V. parvula Bacteroides spp., Eubacterium spp., F. nucleatum, F. periodonticum, Methyl mercaptan from L-methionine Porphyromonas endodontalis Bacteroides gracilis, B. intermedius, B. loescheii, B. oralis, Eubacterium lentum, Eubacterium spp., F. nucleatum, Mitsuokella dentalis, Hydrogen sulfide from serum Peptostreptococcus magnus, P. micros, P. prevotii, P. propionicum, Porphyromonas gingivalis, T. forsythia, Treponema denticola, V. parvula Methyl mercaptan from serum P. endodontalis, P. gingivalis, T. denticola

Many studies showed that bacterial diversity in the group of patients with IOH is much higher than in the control group. Furthermore, many publications draw attention to the correlation between halitosis and individual microorganisms. The relationship between tongue bacterial composition structure and VSC gases is also mentioned by many authors [3,108]. Many oral bacteria that cause IOH contain similar enzymes. These enzymes are proteins encoded by related genes (megL, lcs, mgl) in the genomes of various bacterial species. The main enzymes are methionine γ-lyase, L-cysteine desulfhydrase, and L-methionine α-deamino-γ-mercaptomethane-lyase [109]. Veloso et al. mentioned that in 85% of the patients IOH is caused by Gram-negative bacteria [6]. According to Wei et al., the oral microbiota responsible for IOH includes a wide range of microbial communities, including 13 phyla, 23 classes, 37 orders, 134 genera, 266 species, and 349 operational taxonomic units. The largest percentage amongst the oral cavity microorganisms are genera, like Prevotella, Alloprevotella, Leptotrichia, Peptostreptococcus, and Stomatobaculum. These bacteria present a higher percentage of occurrence in the sample of patients with IOH than in the control samples from healthy patients [103]. In turn, the presence of bacteria, such as Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria, was demonstrated in both the samples from examined and control groups. Firmicutes was the most abundant phylum in saliva samples from both groups [110,111]. The composition of the tongue microbiota has an essential influence on IOH. The most common molecular technique for testing and evaluating an oral cavity is the sequencing [5,107,112,113]. Seerangaiyan et al. published a review in 2017, in which they showed the composition of the bacteria of Aggregatibacter, , Capnocytophaga, Clostridiales, Leptotrichia, Parvimonas, Peptostreptococcus, , Prevotella, Selenomonas, Dialister, Tannerella, and Treponema in the group of patients with IOH. Using the amplification of 16S rRNA, the researchers also demonstrated a high prevalence of Solobacterium moorei strains in the IOH group. By testing the control group, significant differences were found in both groups of healthy and sick people. Furthermore, using polymerase chain reactions (PCRs), Seerangaiyan et al. showed the positive correlation of Leptotrichia spp. and Prevotella spp. to oral malodor severity, contrary to Haemophilus spp., Gemella spp. and Rothia spp. [5]. Patients with IOH have a specific biofilm on the dorsal part of the tongue. Bernardi et al. stated that this biofilm consists of a significant proportion of Fusobacterium nucleatum and Streptococcus spp. J. Clin. Med. 2020, 9, 2484 9 of 17

The occurrence of these two types of bacteria in patients with IOH was completely related. According to the authors, these microorganisms contribute significantly to IOH and can be treated as treatment targets [114]. In other research, Bernardi and partners showed that Actinomyces graevenitzii and Veillonella rogosae were closely related to the occurrence of IOH in a group of volunteers. Also, /oralis, S. pseudopneumoniae, and S. infantis, as well as Prevotella spp. were detected often in malodor patients. Moreover, following the earlier findings, the researchers’ results revealed the presence of Actinomyces odontolyticus, Solobacterium moorei, Prevotella melaninogenica, Fusobacterium periodonticum, and Tannerella forsythia in IOH patients. Furthermore, microorganisms such as Streptococcus parasanguinis, S. salivarius, Veillonella spp., and Rothia mucilaginosa dominated in the oral microbiota of healthy people [112]. Yitzhaki et al. noticed the connection between IOH and wearing dentures. The unpleasant odor was organoleptically assessed and the oral microbiome was analyzed using Next Generation Sequencing 16S rDNA technology. Researchers have identified bacterial taxa, including nine phyla, 29 genera, and 117 species. The samples taken from patients with IOH showed the dominance of the phyla Firmicutes and Fusobacteria and the genera Leptotrichia, Atopobium, Megasphaera, Oribacterium, and Campylobacter. The analyses revealed a significant diversity of the oral microbiota among samples from IOH patients wearing alveolar dentures and significant differences in comparison to the control group [113]. The use of tobacco also has a huge impact on the oral microbiota diversity. After examining a group of smokers and non-smokers, researchers reported that in both groups, most of the oral microbiota were Gram-negative bacterial strains. Simultaneously, Klebsiella pneumoniae dominated in smokers’ saliva and aeruginosa in non-smokers’ saliva samples. An essential finding of the research was also that the Candida species accounted for the largest percentage of microbes amongst smokers with halitosis [97]. Al-Zyound et al. performed tests showing an increased level of three bacterial genera in smokers: Streptococcus, Prevotella, and Veillonella. Researchers provided evidence that tobacco smoking has a direct effect on the oral microbiota. They also suggested that after smoking cessation, it is possible to return to the standard composition of the oral cavity microbiota [115]. Wu et al. noticed significant changes in the oral microbiota that occurred amongst obese people suffering from malodor. The Prevotella, Granulicatella, Peptostreptococcus, Solobacterium, Catonella, and Mogibacterium were more abundant genera in the obesity group than in healthy persons [116]. Halitosis has often been reported amongst the symptoms related to Helicobacter pylori and gastroesophageal reflux disease. Anbari et al. made the observations that the incidence of malodor amongst Helicobacter pylori-positive patients was 74% [2]. However, Tagerman et al. disagreed about a possible relationship between Helicobacter pylori infection and objective halitosis [22]. It is difficult to identify bacteria that promote malodor in children. The most common groups of oral bacteria in children with IOH are Veillonella spp., Prevotella spp., Fusobacterium spp. However, there is no difference in the abundance of these microorganisms in children with IOH and those without [110]. In Table3, results of studies concerning microbiota associated with IOH are presented. Summarizing the table, the oral bacteria that are most related to IOH are Actinomyces spp., Bacteroides spp., Dialister spp., Eubacterium spp., Fusobacterium spp., Leptotrichia spp., Peptostreptococcus spp., Porphyromonas spp., Prevotella spp., Selenomonas spp., Solobacterium spp., Tannerella forsythia, and Veillonella spp. J. Clin. Med. 2020, 9, 2484 10 of 17

Table 3. Results of studies concerning bacteria associated with intra-oral halitosis (IOH).

Bacteria Related to Intra-Oral Halitosis Studied Population Study Method Reference Bacteroides gracilis, B. intermedius, B. loescheii, B. oralis, Capnocytophaga ochracea, Centipeda periodontii, Eikenella corrodens, Eubacterium brachy, E. lentum, E. limosum, Fusobacterium alocis, F. nucleatum, F. periodonticum, F. sulei, Mitsuokella dentalis, Peptostreptococcus anaerobius, P. magnus, P. micros, 9 persons Bacterial culture [96] P. prevotii, Porphyromonas endodontalis, P. gingivalis, Propionibacterium propionicum, Selenomonas artemidis, S. dianae, S. flueggei, S. infelix, S. noxia, S. sputigena, Tannerella forsythia, Treponema denticola, Veillonella dispar, V. parvula Fusobacterium sp., P. gingivalis, 16 IOH adults or children Bacterial culture [117] Campylobacter rectus, F. nucleatum, P. micros, 40 IOH patients Anaerobic culture [118] P. gingivalis, P. intermedia, T. forsythia Fusobacterium sp., P. gingivalis, P. intermedia, 20 IOH adults Anaerobic culture [119] T. forsythia P. gingivalis, P. intermedia, P. melaninogenica, checkerboard P. nigrescens, Streptococcus constellatus, T. forsythia, 10 adult persons DNA-DNA [120] T. denticola, V. parvula hybridization technique Actinomyces israelii, A. neuii, A. odontolyticus, Aggregatibacter actinomycetemcomitans (serotype a), Checkerboard Atopobium parvulum, Prevotella bivia, P. disiens, 21 IOH adults DNA-DNA [121] P. nigrescens, , Staphylococcus hybridization epidermis, S. constellatus, Streptococcus mitis, T. forsythia, V. parvula F. nucleatum, P. gingivalis, T. forsythia 30 adults PCR [122] P. gingivalis, P. intermedia, T. forsythia 101 IOH adults PCR [123] P. gingivalis, P. intermedia, P. nigrescens, T. forsythia, 29 IOH patients and 10 Real-time PCR [124] T. denticola healthy adults Quantitative real-time F. nucleatum, Solobacterium moorei, T. forsythia 78 adult males [35] PCR A. actinomycetemcomitans, F. nucleatum, P. gingivalis, 31 IOH patients and 31 16S rDNA-directed PCR [125] P. intermedia, T. denticola healthy adults Atopobium sp., Dialister sp., Eubacterium sp., Fusobacterium nucleatum, Leptotrichia sp., Megasphaera sp., Neisseria sp., Parvimonas sp., 30 IOH patients and 13 PCR and sequencing [107] Peptococcus sp., Peptostreptococcus sp., P. gingivalis, healthy persons P. endodontalis, Prevotella sp., Selenomonas sp., Solobacterium sp., SR1 sp., Veillonella sp. A. odontolyticus, F. periodonticum, Leptotrichia sp., 6 IOH patients and 6 Okadaella gastrococcus, Prevotella melaninogenica, PCR and sequencing [112] healthy adults S. moorei, T. forsythia phyla Firmicutes and Fusobacteria, genera Atopobium, Campylobacter, Leptotrichia, 26 full dentures patients PCR and sequencing [113] Megasphaera, Oribacterium A. odontolyticus, Atopobium parvulum, Lysobacter-type species, Porphyromonas sp., P. melaninogenica, 20 IOH patients and 12 PCR and DNA [126] P. pallens, P. veroralis, Streptococcus salivarius, S. mitis, healthy adults sequencing S. oralis, V. parvula Eubacterium sp., Dialister sp., Granulicatella elegans, 8 IOH patients and 5 PCR and DNA Porphyromonas sp., P. intermedia, Staphylococcus [127] healthy adults sequencing warneri, S. moorei Aggregatibacter sp., A. segnis, Campylobacter sp., Capnocytophaga sp., Clostridiales, Dialister sp., 16 IOH patients and 10 Leptotrichia sp., Parvimonas sp., Peptostreptococcus sp., 16S rRNA sequencing [5] healthy adults Peptococcus sp., Prevotella sp., Selenomonas sp., SR1, Tannerella sp., TM7-3, Treponema sp. J. Clin. Med. 2020, 9, 2484 11 of 17

Table 3. Cont.

Bacteria Related to Intra-Oral Halitosis Studied Population Study Method Reference Prevotella sp., Leptotrichia sp., Actinomyces sp., 5 IOH children and 5 Porphyromonas sp., Selenomonas sp., Selenomonas noxia, 16S rRNA sequencing [128] healthy Capnocytophaga ochracea A. parvulum, Eubacterium sulci, F. periodonticum, 6 IOH patients and 5 16S rRNA sequencing [129] Dialister sp., S. moorei, Streptococcus sp., TM7-8, healthy adults A. odontolyticus, Hemophilus parainfluenzae, Gemella 16S rDNA amplicon sp., Leptotrichia wadei, Prevotella tannerae, Streptococcus 29 adults [130] sequencing sp., 16S rRNA gene Actinomyces sp., Prevotella sp., Veillonella sp. 10 adults [131] sequencing Aggregatibacter sp., Anaerovorax sp., , Butyrivibrio sp., Dialister sp., Eikenella sp., Mogibacterium sp., Moraxella sp., Peptococcus sp., 40 IOH adults 16S rRNA sequencing [132] Peptostreptococcaceae, RF39, Tannerella sp., Treponema sp., Streptococcus halitosis sp. nov. strain VT-4 - 16S rRNA sequencing [133]

5. Conclusions The IOH is formed by volatile compounds, among which volatile sulfur compounds (VSCs), such as hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, and methyl mercaptan, are predominant. VSCs are produced mainly by anaerobic bacteria belonging to genera Actinomyces, Bacteroides, Dialister, Eubacterium, Fusobacterium, Leptotrichia, Peptostreptococcus, Porphyromonas, Prevotella, Selenomonas, Solobacterium, Tannerella, and Veillonella. A combination of different microbial techniques is recommended to analyze the etiological microflora associated with IOH. Increased knowledge of the microbiota of the oral cavity and especially tongue biofilm is essential for further research to develop new halitosis therapy strategies.

Author Contributions: Conceptualization, K.H. and T.M.K.; data curation, K.H., M.M.J., Z.Ł.B. and T.M.K.; writing—original draft preparation, K.H., M.M.J. and T.M.K.; writing—review and editing, K.H., Z.Ł.B. and T.M.K.; visualization, T.M.K.; supervision, T.M.K. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by the Chancellor of the Faculty of Pharmacy, PUMS, Lucjusz Zaprutko. Conflicts of Interest: The authors declare no conflict of interest.

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