The effect of lactoperoxidase-thiocyanate-hydrogen peroxide on the metabolism of cariogenic micro-organisms in vitro and in the oral cavity

(1)

THE EFFECT OF

LACTOPEROXIDASE-THIOCYANATE-HYDROGEN PEROXIDE ON THE

METABOLISM OF CARIOGENIC

MICRO-ORGANISMS IN VITRO AND

IN THE ORAL CAVITY

H. HOOGENDOORN

(2)

THE EFFECT O F

LACTOPEROXIDASE-THIOCYANATE-HYDROGEN PEROXIDE ON THE METABOLISM OF CARIOGENIC

MICRO-ORGANISMS IN VITRO AND IN THE ORAL CAVITY

BIBLIOTHEEK TU Delft P 1819 4049

(3)

THE EFFECT OF

LACTOPEROXIDASE-THIOCYANATE-HYDROGEN PEROXIDE ON THE

METABOLISM OF CARIOGENIC

MICRO-ORGANISMS IN VITRO AND

IN THE ORAL CAVITY

PROEFSCHRIFT

ter verkrijging van de graad van doctor in de technische wetenschappen aan de Technische Hogeschool Delft, op gezag van de rector magnificus ir H B Boerema, hoogleraar in de afdelmg der elektrotechniek, voor een commissie aangewezen door het college van dekanen te verdedigen op woensdag 23 oktober 1974 te 14 00 uur door HENDRIK HOOGENDOORN scheikundig ingenieur geboren te Rotterdam

/B/0 ^/OVO

1974

(4)

Dit proefschrift Is goedgekeurd door de promotoren Prof, dr K. G G Konig en

(5)

Aan: Kathe,

(6)

Dankwoord

Mijn buitengewone erkentelijkheid gaat uit naar alien die hebben bijgedragen aan de totstandkoming van dit proefschrift

In het bijzonder geldt dit voor mijn promotoren, Professor Dr K G G Konig van de Katholieke Universiteit Nijmegen en Professor W Berends, Technische Hogeschool Delft

Evenzo gaat mijn dank uit naar de Directie van AKZO, consumenten producten divisie, die dit onderzoek heeft mogelijk gemaakt

Van de velen die op enigerlei wijze aan dit proefschrift hebben bijgedragen wil ik de medewerkers van het AKZO-Research and Development laboratorium in Den Haag met onvermeld laten

Ook de Directie van het Instituut voor Doven, Effatha en de hoofden der scholen dank ik voor de medewerking bij het onderzoek waarbij Drs J v Meel met veel enthousiasme de metingen heeft verncht

(7)

Chapter 1 INTRODUCTION AND SURVEY

OF LITERATURE

1.1. Development and prevention of carious lesions

The first step in the process of caries formation is the primary colonization of bacteria on the tooth surfaces and subsequent formation of dental plaque The tooth surface, being inorganic (though covered with salivary glycoprotein), IS quite different from any other surface of the body, and - together with the saliva - It IS responsible for the selection of the dental flora (Gibbons, 1970 and Rolla, 1970) Only a limited number of bacterial species can survive on a clean tooth surface and initiate plaque

The next step, which occurs mainly on rough surfaces and retention sites, is the massive accumulation of various bacterial cells, residues of cells and bacterial products Bacterial products which enhance plaque formation and determine its physical properties, are extracellular polysaccharides generated mainly from sucrose by certain micro-organisms

When synthesizing these extracellular polysaccharides, the cell uses the residual monosaccharides as a source of energy Moreover the cell has the ability to store polysaccharides intracellularly as a reserve food supply,

therefore even when the environment no longer contains fermentable sugars, the cell still possesses metabolic activity for quite some time.

If the bacteria have no cytochrome system as is the case with streptococci and lactobacilli, the production of energy results in formation of large amounts of acid Micro-organisms present in the plaque convert sugars into lactic acid (and other organic acids), resulting in a substantial decrease of the pH (Geddes 1973)

The drop in pH, in case it is not thwarted by the buffering capacity of the saliva, leads to carious demmeralization in the deeper layers of the tooth because of the better solubility of calcium hydroxyphosphate (its major constituent) in acid environment

The process of demmeralization is rather slow, therefore exposure of dental surfaces to acid food does comparatively little harm, but a rather permanent

(10)

(though not extremely) low pH on the teeth, caused by micro-organisms in the plaque, is destructive This particular situation arises at night as the dental plaque IS not removed mechanically and the buffering capacity of the saliva is minimal because of decreased secretion

Remineralization occurs when the pH returns to normal and the calcium and phosphate ions in the saliva are redeposited to some extent on the locations where solution took place

The thicker the plaque, the more difficult the transport of ions from saliva to the tooth surface gets and due to the lack of neutralizing action of the saliva

near the tooth surface (covered with a thick layer of plaque) low pH values will be reached, resulting in tooth decay

The measures which can be taken to prevent development of carious lesions may be deducted from the etiology

- As carious lesions are formed by acids originating from sugar, the first requirement is to limit the frequency of sugar consumption

- The second measure, which like the first is a matter of education is to remove as much of the plaque from the tooth-surface as possible This is most readily achieved by thorough cleansing of the teeth. This second measure IS very necessary for more than one reason, because plaque is essential in the etiology of caries and the formation of calculus and development of gingivitis

- The third measure to inhibit caries is the use of fluoride

By replacing hydroxyl ions in the enamel by fluoride the solubility is reduced and the tooth becomes less susceptible to acid attack At the same time remineralization of the tooth is promoted

Although it is known that fluoride ions have an inhibiting effect on certain metabolic enzymes in bacterial cells, little is known about the quantitative meaning of this effect.

Compared to these three measures, which have been proven effective in prac-tice all other measures have so far been less successful The application of fissure sealants may prevent the development of caries but requires almost as much work as a preventive filling with standard materials (Silverstone, 1974) Removal of plaque other than by conventional mechanical cleansing has been attempted with enzymes After several vain attempts with proteases amylases and lipases the enzyme dextranase gave some indication that it was able to degrade bacterial polysaccharides Under special conditions the addition of dextranase to the food could be shown to reduce caries formation (Guggenheim et al , 1969)

The w o r k of Guggenheim in particular (Guggenheim and Newbrun, 1969 and Guggenheim, 1970) showed that the so called dextrans formed by Streptococcus mutans were not in fact pure dextrans Their composition varies greatly from strain to strain and is also dependent on the available sugar concentration Besides a - ( l ^ - 6 ) bonds in the molecule there are many a-(1^-3) linkages and

(11)

great differences in the degree of polymerisation and branching

In order to differentiate between these recently detected polymers synthesized by Streptococcus mutans (often erroneously named dextrans) and the classic a - ( 1 - ^ 6 ) linked dextrans, Guggenheim suggested the name mutans for polysaccharides which are mainly built up of a-(1^-3) linkages Due to the variation in composition of these glucans, the same objection for the enzyme mutanase (splitting only a-(1^-3) linkages) as for dextranase is valid Moreover, the time of exposure is so short that effects can only be expected if the enzyme and sucrose are present at the same time Clinical effects have not been l e p o r t e d yet

Reduction in the total flora can in principle be achieved by the use of antibiotic (Fitzgerald, 1972) and bacteriostatic agents The objections to the use of antibiotics over an extended period of time are well known

It can lead to the development of resistent strains which can disturb the natural balance of the oral and intestinal flora

For bacteriostatic agents the same objections are true to some extent, in addition to the fact that most of them are not present in the oral cavity in effective concentrations for more than 1-3 hours

M u c h less IS known about the manner in which the flora recovers and of the metabolic condition of the cells after this period

A prolonged effect in plaque on teeth has been shown for chlorhexidme Apart f r o m the brown discolouration of the mouth, caused by a disturbance of the microbial ecology little is known of any side effects after its continuous use (Flotra et al , 1971) The impression is, however, that a beneficial effect is only achieved during strictly controlled and regular use

1 2 The caries preventive activity of saliva

It IS known that saliva plays a role in the caries process The quantity, t h e buffering action and the calcium and phosphate content of saliva are all factors which protect teeth against demmeralisation due to bacterial acid formation The importance of antibacterial factors in saliva such as lysozyme and the antibacterial system based on lactoperoxidase has never been

com-pletely clarified The activity of the enzyme lysozyme is limited to a group of micro-organisms which are not normally found in the oral flora One could assume that this is only the case because these organisms cannot establish themselves m the mouth in the presence of lysozyme On the other hand the antibacterial system consisting of lactoperoxidase, thiocyanate and hydrogen-peroxide works mainly against lactobacilli and streptococci as shown by in vitro experiments Both the sensitive lactobacilli and the less sensitive oral strep-t o c o c c i (Morrison, 1968) are presenstrep-t in strep-the moustrep-th in large numbers, which_ IS not the case for those organisms against which lysozyme is active

(12)

it was concluded that the lactoperoxidase system would not work adequately It was assumed that the flora in the plaque is separated from the saliva and

that no interaction between the two occurs

From this background it is understandable that the stimulus was lacking to continue investigations into the role of lactoperoxidase in the ecology of the mouth

1.3. Lactoperoxidase as a bacteriostatic factor in saliva

1 3 1 Discovery in milk and saliva

The presence of peroxidase in milk was shown as early as 1881 by Arnold In 1932 Elliott isolated the enzyme in a fairly pure form Originally it was thought that all peroxidases were identical, but the work of Theorell (1943) proved that the peroxidase in milk, thereafter named lactoperoxidase, had its own special characteristics

In 1909 MacDonald and Smith reported that there was an enzyme in saliva which exhibited both "oxidase and peroxidase" properties For some time it was assumed that this was the result of products originating from white blood cells untill Mosimann and Summer (1951) showed this activity to be present in pure saliva After it appeared that there were great similarities between peroxidase in milk and in saliva it was shown by Morrison et al in 1965 that they are identical From these investigations differences between lactoperoxidases of different animals were found (Morrison, 1968). Though these differences were small, it nevertheless seemed necessary to report the source of any lactoperoxidases used

The first to mention the antibacterial effect of constituents of milk was

Hanssen in 1924 The material called lactenine was intensively studied by Jones et al in the years 1927-1930 From this work, later confirmed by Wilson and Rosenblum (1952), it appeared that the inhibitory effects on lactic acid forming bacteria varied tremendously

Although the inhibition was stronger for streptococci belonging to the Lance-field groups A, F and G, the variations within the groups were large

The investigation of antibacterial substances in saliva was continued by Dold and Weigman (1934) They showed that this material, called inhibme, was not the same as the lysozyme found by Fleming (1922) In 1947 it was shown by Thompson and Johnson that the salivary antibacterial effects could be correlated with the formation of hydrogenperoxide Their conclusion was, that the inhibiting factor was hydrogenperoxide

In the following years the bactericidal effect of saliva was investigated by many workers, who concentrated mainly on the inhibition of lactobacilli (Clough, 1935, Hill, 1939, van Kesteren et al 1942, Kerr and Wedderburn 1958, Zeldow, 1959)

(13)

A third name for the system was introduced in 1960 by Matsumura, who called it the Salivary Antilactobacillus Factor (the S A - f a c t o r ) This name is prin-cipally used in the Japanese literature

The investigation by Wright and Tramer (1958) proved that the bacterial inhibition due to saliva and milk was the result of the combined working of the enzyme lactoperoxidase, and the thiocyanate ion It was shown by Dogon et a l . (1962) and Zeldow (1963) that the iodide ion can function as cofactor instead of the thiocyanate ion

Many workers have studied the role of thiocyanate as a cofactor (Stadhouders and Veringa, 1962, Jago and Morrison, 1962, Oram and Reiter, 1966, Hogg and Jago, 1970)

It was proposed that following oxidation of the thiocyanate ion some component was formed which exhibited the observed inhibiting effect

As It proved impossible to show an inhibiting effect when this system was separated from the bacterial cells by a dialysis membrane it was assumed that the active component has a low stability On the other hand it appeared to be possible to keep the components of the inhibiting system together for several hours, without loss of activity It was suggested by Steele and Morrison (1969) that the effect of the thiocyanate ion was purely protective, preventing the mactivation of the enzyme Inhibition should occur by formation of some bond between enzyme, thiocyanate ion and the cell, allowing the hydrogenperoxide to be transferred directly to the cell No satisfactory explanation has yet been found to account for the observed phenomena Based on the relative concentrations of thiocyanate and iodide ions found in milk and saliva it can be assumed that the inhibiting factor is due to the lactoperoxidase-thiocyanate combination

Therefore, the many publications of Klebanoff et al using iodide ion as cofactor, will not be considered here Nevertheless it is of interest to notice that (with iodide ion as cofactor) a broad spectrum of bactericidal activity and even virucidal, fungicidal and spermicidal activity can be shown

It has been mentioned already that lactobacilli can be effectively inhibited

This was shown for Lactobacillus casei A T C C 4646 (Dogon, 1962), Lactobacillus acidophilus A T C C 4357 (Zeldow, 1963) and Lactobacillus plantarum

A T C C 8014 (Iwamoto, 1966).

On the other hand the inhibition of streptococci varied from strain to strain. confirming earlier work by Jones (1930), Stadhouders showed good inhibition of Streptococcus cremoris strain 972, though strain 803 was hardly inhibited Mickelson (1966) found that Streptococcus agalactiae could adapt to oxidative metabolism and, in that case, was not affected by the lactoperoxidase system. Four strains of Streptococcus mutans were investigated by Morrison (1968). These exhibited little or no inhibition when exposed to the lactoperoxidase system Very little investigation of pathogenic micro-organisms has been carried out. It appeared from the w o r k of Hanssen (1924) and Weigman (1934) that salmonella species and Corynebacterium diphtenae might be sensitive to the lactoperoxidase system

(14)

1 3 2 Biological properties of lactoperoxidase

It has been shown by Oram and Reiter (1966) that in Streptococcus cremoris

strain 972 the enzyme hexokinase is inactivated by the lactoperoxidase system

In the case of the less sensitive more aerobic strain 803 there is only a reduction in the activity of hexokinase They discovered a positive correlation between the resistance of strain 803 and the possession of a NADH-oxidising factor in these cells

Inactivation of lactoperoxidase by micro-organisms was investigated by

Kiermeyer et al (1960-1966) as well as by Pickering et al (1962) They found that certain organisms could inactivate lactoperoxidase, using hydrogen-peroxide generated by cell metabolism This inactivated lactoperoxidase was chemically active, but m biological experiments it appeared to have lost its antibacterial function

It was shown by Austin (1961) that the production of lactoperoxidase by the salivary glands began 5 to 7 days after birth Morioka (1969) showed that this was also the case for germ free rats So the enzyme lactoperoxidase appears not to be induced by the development of the oral flora

A remarkable finding was, that in colostrum, the concentration of lactoperoxidase IS very high and after 1 to 2 weeks returns to a level which is normally found in milk

Therefore the new born acquires the antibacterial system from its mother's milk, later from its own saliva

1 3 3 The chemical properties of lactoperoxidase

The enzyme lactoperoxidase is a true peroxidase containing one haemine group per molecule It was found that 6 different fractions can be obtained from milk, which can be divided in two groups, A and B

The ammo acid composition in both groups was found to be almost the same, however a lower carbohydrate content was found in group A

The molecular weights were approximately 76 500 and 78 500 for group A qjid B respectively The differences within these groups may be due to deamination of asparagine and glutamine residues (Carlstrom, 1969)

The enzyme forms complexes with cyanide, carbonmonoxide, pyridine and fluoride and forms the same series of compounds with hydrogenperoxide and monoalkylhydroperoxides as does horseradish peroxidase Lactoperoxidase catalyses the oxidation of most of the usual hydrogen donors (e g pyrogallol, ascorbic acid, ferrocytochrome C, guaiacol, benzidine, p-phenylenediamme), and IS able to oxidise dihydroxyfumarate, dihydroxyphenylalanine and thiocyanate However, as soon as the hydrogenperoxide concentration exceeds a certain level, mactivation of the enzyme will occur (Morell, 1953)

(15)

The Michaelis constant was found to be 1 08 10"' M (hydrogenperoxide-pyrogallol)

The investigations of Hultquist and Morrison (1963) showed that the prosthetic group can be cleaved from the enzyme by different methods, a.o by means of hydrazine in acetic acid They showed that the heme prosthetic group is bound to the tetrapyrrol nucleus with two double bonds and one or more hydroxyl groups to the side chain

Chemically lactoperoxidase differs in two ways from other peroxidases Firstly the enzyme has a high carbohydrate content (approximately 10 percent) and secondly there is an ester bond between the apoprotein and the prosthetic group, this results in a very stable enzyme Oxidation of the enzyme can result in denaturation at low temperatures

The mactivation of the enzyme by streptococci is the result of the formation of hydrogenperoxide and the subsequent oxidation of protein in the enzyme (Kiermeier et al 1960-1966, Pickering et al 1962)

The mechanism of oxidation of peroxidase by hydrogenperoxide and the follow-ing reactions are given below (Saunders et al. 1964)

Peroxidase •<• > H2O2 > Compound I Com H2O2 > C o m | Comf

Peroxidase reacts with one molecule of hydrogenperoxide to give compound I This compound is reduced in two steps in each of which one electron is ac-cepted Compound II however can react in two ways neither of which gives rise to the original peroxidase If a second molecule of hydrogenperoxide reacts with compound II, a compound III is formed which changes to compound IV with disintegration of the haemine centre

By means of catalase, compound III can be converted to compound II Thus catalase prevents tbis particular form of mactivation

A second side reaction (not shown m the figure) is the spontaneous transformation of compound II into compound I (Maguire, 1972), or even to the ground state, such as happens with horseradish peroxidase In this case the enzyme protein

< 1e r-v 1e > Donor )ound II ound III

i

ound IV (inactive)

(16)

acts as the donor and this gives a peroxidase with different properties The velocity of catalysis is greatly decreased in this case and the inhibiting effect IS lost. Most of the experimental work on peroxidase systems is carried out using these modified forms because the longer reaction times are easier to study The spontaneous decay of compound II occurs when there is no electron donor present As thiocyanate ion can act as an electron donor it protects the lacto-peroxidase against this form of mactivation

1 3 4 The lactoperoxidase system and dental caries

Although little has been published concerning any direct relationship between lactoperoxidase and caries, several interesting related articles are known The earliest publication was in 1908 Based on a clinical test on 300 people. Beach concluded that few caries lesions occurred in people who had a high thiocynate ion concentration in their saliva He stated that in general the thiocyanate ion had a favourable influence on oral health In the following years (1910-1914) Gies et al made an exhaustive investigation of the effect of the thiocyanate ion on the oral flora They found that the concentration in the mouth was too low to have any effect As this investigation was earned out in vitro the enzyme lactoperoxidase was not present and positive results could not have been

expected As a result of this publication the interesting results obtained by Beach were disregarded

In later years Clough (1935) and Hattyasy (1965) investigated the relation between the antibacterial effect of saliva and dental caries Clough studying the inhibition of bacterial growth on plates, found no relation with caries but Hattyasy did find a correlation, measuring the action of saliva on the production of acid by oral bacteria The number of cavities, fillings and missing teeth (DMFT-lndex) wasr used by these workers to classify their subjects as caries active or caries inactive

This classification could be affected by a period of high caries activity in the past or a possible sudden change in caries activity during the investigation A number of publications by Dold and Weigman (1934-1942) concerning the antibacterial working of saliva should be mentioned They reported only the pres-ence of an antibacterial system in the mouth and made no mention of possible role in the prevention of caries

The publication of Wolfe and Turner (1957) is surprising They found when the result of an investigation on 600 children was statistically analysed that low peroxidase activity correlated with few carious lesions, and vice versa Because the results of ten children could not be included in the statistical analysis applied, these results were omitted Just those ten children had practically no caries and showed little peroxidase activity

Courant (1967) showed that the thiocyanate concentration in saliva is higher for smokers than for non-smokers This higher concentration resulted in an increased antibacterial activity against Lactobacillus acidophilus If, however,

(17)

Lactobacillus case/ was used as test organism no difference was found

The activity of the lactoperoxidase system against Streptococcus mutans was investigated by Morrison (1968) In his experiments four strains were used which had been found to cause caries in animal tests He found that bovine lacto-peroxidase gave a slight inhibition only on strain HS-7 The strains HS-6, HS-10 and FA-1 were not inhibited When pig peroxidase was used, all four strains were inhibited This inhibition also occurred in the absence of thiocyanate, but was somewhat reduced

Summarizing the results of these publications it is not possible to get a clear picture of the effect of lactoperoxidase

Published results are often contradictory, sensitive strains seem to be common in the mouth flora but those selected strains used in some animal experiments happen to be influenced very little by the lactoperoxidase system

(18)

Chapter 2 EXPERIMENTAL

2.1. Aim of the investigations

The presence of lactoperoxidase in saliva suggests that this enzyme may influence oral micro-organisms and thus the ecology of the mouth

The mam aim of this investigation was to determine the effect of lactoperoxidase on the oral flora The results of previous workers suggested that particular lactobacilli and certain strains of streptococci ate inhibited by lactoperoxidase Because these organisms play a role in caries etiology, the relationship was investigated

The investigation consisted of the following parts

1 Determination of inhibition of micro-organisms by the lactoperoxidase system 2 Ranking of micro-organisms on the basis of their sensitivity to the

lacto-peroxidase system

3 Investigation of the three components of the system

4 Investigation of the possible effects of other factors on the working of the system

5 Study of the reaction mechanism

6 Testing the activation of the system in vivo

ad 1 The first question that arises is whether the oral strains are inhibited to any measurable degree by lactoperoxidase This necessitates the comparison of different test methods before coming to a selection of an assay suitable to measure the inhibiting effect

ad 2 Is it possible to rank the micro-organisms according to their sensitivities to inhibition by lactoperoxidase? It was important to establish whether the acid formation in the mouth, which leads to caries formation is caused by micro-organisms which are not inhibited by the lactoperoxidase system

ad 3 If the lactoperoxidase system contributes to the prevention of acid formation then a failure of this system could result in the formation of lesions As the system is composed of three components a lack in each of them could lead to

(19)

inadequate working of the system

- Lactoperoxidase It had to be determined if the level present in the saliva could be the cause of a lack of effectivity of the system

- Thiocyanate ion The possible influence of the thiocyanate ion was reported by Beach (1908) Consideration should be given to the correlation between the thiocyanate ion concentration in both saliva and the diet as great changes in diet have occurred since 1908 and it would be impossible to repeat early results It IS important that the minimum concentration of thiocyanate necessary to give inhibition be determined

- Hydrogenperoxide In contrast to the other two components, hydrogen-peroxide does not originate from the salivary glands, but is produced by the oral flora In this respect there is a difference between the lactoperoxidase systems in milk and in saliva, as hydrogenperoxide m milk is generated by the xanthine/xanthineoxidase system The system in milk is therefore less dependent on the hydrogenperoxide producing micro-organism According to Kraus (1955) approximately 60% of the flora in a healthy mouth forms hydrogenperoxide Any inadequate working of the lactoperoxidase system in the mouth will probably be due to insufficient hydrogenperoxide present

ad 4 Although all three components of the system may be present, some disturbing factors may result in inadequate working The following points in par-ticular are of interest

- Inactivation of the enzyme by denaturation, oxidation or by reaction of some compound with the iron atom of the porphyrme nucleus of the enzyme

The extreme stability of the enzyme has already been mentioned (13 3 ) so that in this case we need only consider the last two modes of mactivation Also the effect of the fluoride ion as reported by Wiseman (1970) should be investigated

- Factors which affect in some other way the effect of the enzyme Other perox-idases than lactoperoxidase may compete with the available hydrogenperoxide Especially catalase may be of importance in this respect

ad 5 The starting point of this investigation was the finding by Oram and Reiter (1966) that mhibitipn of Streptococcus cremoris by the lactoperoxidase system IS accompanied by an mactivation of the glycolytic enzyme-hexokinase.

ad 6 The first five points of investigation were designed to discover the positive and negative factors affecting bacterial inhibition of the lactoperoxidase system The final aim was to make a practical use of the system As the lactoperoxidase system is a part of the oral ecology it is not permissible to simplify greatly the test methods For example the interaction between hydrogenperoxide formation and consumption by mixed flora will be missing from gnotobiotical experiments Even the use of test animals will give difficulties when interpretating the results, as it has been shown that the lactoperoxidase from pigs has a much higher activity than the bovine enzyme

(20)

When all these points are considered it becomes obvious that as much of the in VIVO work as possible should be carried out on human subjects As the lacto-peroxidase system inhibits both acid formation and the growth of certain micro-organisms both pH and plaque-formation can be used as experimental parameters

As far as the gingiva is concerned no negative effects are to be expected

2 2 Materials and methods

2 2 1 Micro-organisms

The following strains were used in the investigations

Streptococcus mutans C 67-1 (de Stoppelaar et al 1969)

Sfreptococcus mutans C 67-25 (de Stoppelaar et al , 1971)

Streptococcus mutans OMZ 176 (Guggenheim, 1968)

Streptococcus mutans MW 25 This strain was isolated from human plaque

and further tested due to the phenomenon that the strain gives a positive reaction with hydrogen peroxide on o-dianisidme plates

Streptococcus sanguis OMZ 9 (Guggenheim, 1968) Streptococcus sanguis 804 (Krasse and Carlsson, 1970)

Streptococcus cremoris 803 Ned Instituut voor Zuivelonderzoek Streptococcus cremoris 972 Ned Instituut voor Zuivelonderzoek Lactobacillus casei ATCC 4646

Lactobacillus plantarum ATCC 8014 Actinomyces viscosus WVU 371

Actinomyces viscosus Ny 1 (van der Hoeven, 1974)

Further a number of streptococci isolated out of human plaque during the investigations These strains could be identified according to the methods discnbed by Guggenheim (1968) and Carlsson (1968) as strains of Streptococcus

mutans, sanguis and mitis

2 2 2 Enzymes, chemicals and culture media Lactoperoxidase (EC 1 1 1 1 7 )

I Supplied by Calbiochem PN = 0 56 (claimed 0 65) Activity 1040 U/mg Specific activity 1100 U/mg

II Prepared by the method descnbed by Elliott (1932) PN = 0 02 Activity of standard solution 1000 U/ml Specific activity 50 U/mg

III As II, but punfied by ion-exchange, absorbed on CM Sephadex C 50 and eluted by a sodiumsulfate gradient from 0 t o 0 5 M PN = 0 67 Activity

(21)

1950 U/ml Specific activity 1700 U/mg

IV Prepared as descnbed by Mornson and Hultquist (1963) PN = 0 55 Activity 1600 U/ml

Horseradish peroxidase (EC 11117) Supplied from Boehnnger, Mannheim Grade II

Catalase (EC 1 11 1 6 ) Supplied from Merck, Darmstadt Derived from

Micrococcus lysodeikticus Activity 2000 U/mg

Glucoseoxidase (EC 1 1 3 4 ) Foodgrade, derived from Penicillium amagakiense Varying in activity 1-3 U/mg

Amyloglucosidase (EC 3 2 1 3 ) Foodgrade, derived from Aspergillus niger. Activity 10 U/mg

The activity unit for enzyme activity is defined as the amount of enzyme which catalyses the degradation of 1 umol of substrate per minute under standard conditions

All other enzymes used for analytical purposes were supplied by Boehnnger, Mannheim

With the exception of Laktobazillus Selektiv agar (Merck, Darmstadt) and Actinomyces Broth ( B B L ) the culture media were supplied by Difco, and prepared as prescribed by the manufacturers

Radioactive materials were supplied by The Radio Chemical Centre Amersham England

Other chemicals used were of reagent grade quality manufactured by Baker, Boehnnger or Merck.

2 2 3 Determination of growth inhibition a On plates

The plates for the test were prepared by adding the test micro organism to liquid agar at 45° The number of micro-organisms used was such as to give a concentration in the plate of some 10^ cells per ml

Cells used were preferably in the stationary growth phase After the agar had set, holes with a diameter of 8 mm were bored Unless otherwise stated these holes were filled with 0,1 ml solution of 2 mM thiocyanate, 0 03 mM hydrogenperoxide and a lactoperoxidase activity of 8 units Where the media contained no thiocyanate ions the concentration of the added solution was increased to 20 mM, in other cases 2 mM solution of thiocyanate was added to the medium

The inhibition zones were measured after 18 hours incubation at 30° or 37° b In liquid media

These tests were carried out in test tubes, containing 10 ml medium to which glucose and thiocyanate were added to give 0 1 M and 3 mM respectively These tubes were inoculated with a 24-hour culture (end of the logarithmic growth) giving a viable count of 10^ per ml

(22)

The enzyme concentrations used were, lactoperoxidase (II) 25 U / m l , and catalase 1000 U / m l , and the incubation temperature was 30"^ or 37°.

2 2 4 Inhibition of acid formation

a In test tubes, without pH correction

The ability of cellsuspensions, 10' viable counts per ml, 24-hour cultures, to form acid from glucose was measured by noting the change in pH

The tests were carried out in test tubes, with 10 ml of media and an incubation temperature of 3 7 ° .

b In the cariotron

The cariotron is a device described by Nolte and Arnim' (1964) which is suitable to study the metabolism of micro-organisms when tightly packed in a form like dental plaque The apparatus is a continuous flow microchamber in which a few microliters of a bacterial culture can be packed between bacterial filters On the outside of the filters fluids can be supplied or removed, and there IS space for a pH electrode The sample was prepared by centrifuging 18 hour cultures to get a cell concentration of 1 0 " cells per ml

On one side of the filter the test solution was supplied and the change in pH was measured by two combined electrodes, one on each side of the culture

The instruments and electrodes 7 Gr 221/100 L were supplied by Electrofact In this way the pH changes in a 0 1 M sugar solution flowing along the filter could be measured for all cell suspensions, before and after they had been exposed to the lactoperoxidase system

c Inhibition at constant pH

The acid formation in a 50 ml suspension, containing 2 10^ germs per ml, was measured using a Metrohm combititrator 3D In all cases two parallel tests were run, the compound under investigation being omitted in the blank The tests were performed at 35° and at pH 7 0

2 2 5 Determination of the enzyme activity in cell free extracts

In these test cells which had been cultured for 18 hours in Brain Heart infusion broth were used These cells were harvested, washed and suspended in a phosphate buffer solution (0 1 M, pH 7 0), to give a concentration of

ap-proximately 5 10' germs per ml Where it was necessary to use cells with a low metabolic activity in order to obtain optimal inhibition, the cells were incubated in this buffer for 30 minutes at 37°

For the test 5 ml of the cell suspension was added to the solution of the materials under investigation to give a total volume of 10 ml The resulting suspension

(23)

was incubated for 10 minutes at 37° The suspension was then cooled to 0° and sonified ten times, for periods of 1 minute with a Branson sonifier

B 12, 20 000 Hz, 80 W

The cells and cell fractions in the resulting suspension were separated from the solution by centrifuging, 10 minutes at 25 000 x g in the Sorvall superspeed centnfuge SS-1.

The enzymes in the solution were determined as described by Bergmeyer, 1962. The measured activities were based on the protein content as determined, ac-cording to the biuret method (Perkin Elmar double beam spectrophotometer 124)

2 2 6. Investigation of glycolysis of ^^C-labelled glucose or sorbitol

The preparation of the cell suspension was carried out as described in section 22 5

To 5 ml of the suspension 4 ml of the solution under investigation were added After incubation, 1 ml of a 0,3% ^''C-labelled glucose or sorbitol solution was added (specific activity 16,7 //Ci/mg)

This suspension was incubated for 10 minutes at 37" then cooled to 0° after which the cells were centnfuged (5 minutes at 4000 x g)

This solution was examined to determine if any lactic acid had been formed, by means of thin layer chromatography on MN-cellulose The cell fraction washed with phosphate buffer (0 01 M, pH 7 0, 0°) was added to 5 ml of an acetone-butanol-water solution (1 1 18), immediately heated to boiling and kept at this temperature for 10 minutes The mixture was centnfuged at lOOOOxg and

10 jn\ of the supernatant examined using thin layer chromatography

The TLC plates were then developed twice in the same direction with isopropanol-25% ammonia solution and water (6 1 3) The plate was covered with Agfa-Gevaert Osray T-4 X-ray film which was developed after an exposure of 7 to 14 days

The spots were identified by means of known intermediates and were found to be in accordance with the findings of Stegmeier, 1973

2 2 7 The detection of hydrogenperoxide

- The production of hydrogenperoxide by micro-organisms was observed when plating them out on brain and heart infusion agar containing horseradish peroxidase (HPO) and o-dianisidine (ODA)

The medium for the ODA-plates was made in the following way 5 ml of a solution containing 0 6% ODA and 0 04% HPO (Boehnnger grade II) was passed through a sterile membrane filter (Sartonus) and added to 100 ml of sterilized Brain Heart infusion agar (cooled down to 45°)

(24)

Using this medium (which is a modification of the medium used by Whittenbury) one can detect the hydrogenperoxide forming colonies by their distinct dark brown colour

- The amount of hydrogenperoxide produced by micro-organisms cultured in a liquid medium was estimated by measuring the increase in extinction at

436 nm, due to the peroxidase catalysed oxidation of o-dianisidine by hydrogen-peroxide

After removing the cells by centnfugation, the supernatant culture broth (if necessary diluted to give a measurable extinction) was mixed with 0 5 ml of 0 2 M phosphate buffer pH 7 containing 0 0 1 % ODA and 0 001% HPO (grade II), to give a final volume of 3 ml The increases in extinction at 436 nm due to known concentrations of hydrogenperoxide were compared with the increase due to the hydrogenperoxide in the liquid medium

2 2 8 Measurements of pH on the surface of teeth in vivo

The pH which can be measured on tooth-surfaces depends on a number of factors

- Firstly, the composition and the metabolic condition of the flora

- Secondly, host factors such as buffering capacity and quantity of saliva In order to obtain results which may give some information about the oral flora ;n situ, a number of factors had to be standardized For this reason the

measurements were earned out as follows

a Whilst flora composition on the tooth varies from place to place, but on each spot IS rather constant in time, the measurements were taken on exactly defined locations

b The comparative measurements were taken at the same time each day In all cases they were at least 172 hours after the meal

c The measurements were carried out by the same person and the readings were taken by another, but always the same person A third person paid attention to the administration and the control of time schedule

d All measurements were made in dupIo (first upper molars left and right) and repeated after consumption of sugar This measurement was done exactly 10 minutes after the start of sugar consumption

e In some testsenes sugar was consumed by means of sweets weighing approximately 5 grams and in other tests by means of a rinse with 60% sucrose dunng 60 seconds

f The apparatus used was calibrated before, during and after each testsenes

(performed on approximately 20 subjects)

The pH measuring apparatus consisted of a pH-meter type 53A and an electrode system designed according to Thompson and Brudevold, 1954 all supplied by Electrofact, Amersfoort, Netherlands

This electrode system registrated ± 160 mV between pH 4 0 and 7 0, (53 mV per pH) Readings were taken in mV with an accuracy of 3 mV

(25)

2 2 9 Plaque formation test

Fifteen healthy girls aged 18 years from the Dental Nurse School at the Dental School in Malmo took part in the study They were divided into three groups X, Y and Z with five girls in each Each of these three groups received three different experimental treatments (I II and III) in a randomized block design (latin square)

During the experimental periods each covering five days, the test subjects were instructed as follows

Treatment I

a No active oral hygiene to be performed

b 1 lump of sugar to be eaten every hour ( = 40-50 g / d a y )

c Three daily one-minute mouthrmses (07 30 12 30 and 19.30) with 10 ml of a solution of

Methyl p-hydroxybenzoate 0 1 % Propyl p-hydroxybenzoate 0 0 1 %

Treatment II

b 1 lump of sugar to be eaten every hour ( = 40-50 g / d a y )

c Three daily one-minute mouthrmses (07 30, 12 30 and 19 30) with 10 ml of a solution of

Methyl p-hydroxybenzoate 0 1 % Propyl p-hydroxybenzoate 0 0 1 %

Amyloglucosidase Batch AM-20 1 mg = 15 U Glucoseoxidase Batch Go-73 3 mg = 1 8 U (Go-73 contains catalase)

Treatment III

b No instruction regarding in-between meals were given c No rinses were prescribed

At the start of every penod the following procedures were performed a Staining of the dental plaque with 0 1 % basic fuchsin

b Colour photographs were taken of the teeth

c The teeth were thoroughly cleaned by a dental hygienist until no plaque or stamings were present

d Detailed instructions were given how to behave during the experimental period

At the end of every penod (five days after start) the following procedures were performed

(26)

a Dental plaque was scored using the Plaque Index described by Silness and Loe (1964)

b Staining of the dental plaque with 0 1 % basic fuchsin c. Colour photographs were taken of the teeth

d The teeth were thoroughly cleaned by a dental hygienist until no plaque or stamings were present

e The girls were instructed to perform their normal oral hygiene until the start of the next experimental period three days later

The study was performed as a true double-blind clinical test

2 2 10 Short-term caries trial

After oral examination and voluntary acceptance twenty two female dental assistants, mean age 22 years, were selected to take part in the study Individuals with gingival retractions, pathologically deepened periodontal pockets and a large number of buccal restorations were excluded

Preexperimental period

The participants were scored according to the Plaque Index (PLI) (Silness and Loe 1964) and Gingival Index (Gl) (Loe and Silness 1963)

They had their teeth scaled and polished until no plaque was seen after staining with basic fuchsin Instruction in toothbrushing and the use of wooden tooth-picks twice a day was given During a four week period the teeth of the participants were polished twice a week and a check was made that the oral hygiene was adequately performed At the end of this preexpenmental period the PLI and Gl had approached zero

Experimental penod

On day 0 of the experimental period the PLI and Gl of all tooth surfaces were recorded and the teeth were carefully scaled and polished

Then the buccal surfaces of the following teeth were scored according to a modified Caries Index System originally proposed by von der fehr (1961), premolars and canines in the upper and lower jaws and incisors in the upper jaw. The cervical part of each buccal tooth surface was examined in a binocular dissection microscope equipped with a spotlight (magnification 16x)

Each surface was divided into three segments, one distal, one central and one mesial, which were scored separately The reproducibility of the caries examination was determined by double registration of 210 segments The repro-ducibility was 94 per cent

The Caries Index Score for an entire buccal tooth sur.face was represented by the highest separate score registrated in the segments of the surface The twenty-two individuals were then divided into two groups according to a stratification based on past caries experience from the examination mentioned above At the start of the experim,ent the test group had a mean number

(27)

of 6 2 intact surfaces and 27 7 intact segments The control group showed a mean number of 5 8 intact surfaces and 26 1 intact segments

From then on all participants refrained from all active oral hygiene during the following 21 days The individuals of both groups rinsed their mouths ten times a day with 10 ml of a 65 per cent sucrose solution during two minutes The rinsings were performed with an interval of one hour No restrictions of

diet were prescribed The individuals in the test group also rinsed with 10 ml of a solution containing the enzymes amyloglucosidase and glucoseoxidase during 2 minutes three times a day These rinsings were timed between two sucrose rinsings and were performed in the morning, at noon and in the

evening The individuals in the control group rinsed with the same frequency with a placebo solution. The compositions of the solutions were as follows Test solution

Methyl p-hydroxybenzoate 0 1 % Propyl p-hydroxybenzoate 0 0 1 % Na2HP04l2 aq 7 2 mg NaH2P04l aq 2.8 mg

Amyloglucosidase Batch Am-25 1 mg = 15 U pH 7 0 Glucoseoxidase Batch Go-73 3 mg = 1 8 U

(Go-73 contained catalase) Placebo solution

Methyl p-hydroxybenzoate 0 1 % Propyl p-hydroxybenzoate 0 01 %

Na2HP04l2 aq 7 2 mg pH 7 0 NaH2P04l aq 2.8 mg

PLI and Gl were scored after 7 14 and 21 days After this final examination the teeth v\ere polished and the buccal surfaces were examined for caries in the same way as at the start of the experimental period

Postexperimental period

After the 21 days experimental period the teeth were covered for 10 hours with a fluonde varnish (Duraphaf®, Woelm, Eschwege W G ) The oral hygiene measures were reinstituted, supplemented by daily toothbrushing with 10 ml of 0 2 per cent sodium fluoride solution Six weeks later at the end of the hygiene period, the participants were scored again according to PLI, Gl and Caries Index

All plaque and gingival scorings were performed by one person, as also was the canes examination The study was performed as a true double-blind test

(28)

2.3. Specific experiments and results

2 3 1 The inhibitory action of the lactoperoxidase system

2 3 11 Inhibition of growth

The inhibition due to the lactoperoxidase system was determined using the plate method (section 2 2 3 a ) Investigation was carried out w i t h four media and lactobacilli which are known from literature to be more sensitive to inhibition than oral streptococci (section 1 3 1) The results of some specific experiments are given in table 1 Furthermore it appeared that both the growth conditions and the addition of various materials in low concentrations can greatly influence the results

Table 1 Survey of the inhibition of some micro-organisms forming lactic acid by the lactoperoxidase system determined with different media on plates

Culture medium

Testorganism Lactobacillus Lactobacillus Tryptic Brain-Heart agar Merck agar Difco soy agar Infusion agar Lactobacillus casei Lactobacillus plantarum Streptococcus mutans C 67-1 Streptococcus mutans MW 25 Streptococcus sanguis OMZ 9

-I- -I- Inhibition zone 2 mm or more, very reproducable + Inhibition zone 1-2 mm very reproducable ± Inhibition approximately 1 mm low reproducability

0 Micro-organism does not grow on the medium — No inhibition

The plate method only shows if the bacteria are inhibited in the g r o w t h phase It does not give any indication if there has been an extended lag-phase To measure this effect an investigation was carried out in liquid media The results are given in figures 1-3

I -0 0 0 — -f--1- + -t-

(29)

-1-pH 6 5 * ^^"'*~"*—-, ^ \ ' • ^ ~ • /

~t--Brain Heart Infusion broth

pH

Tryptic Soy broth

Lactobacillus broth, Difco

2° ^Tmolhourt)

Brain Heart Infusion broth

Tryptic Soy broth

^ ..Tlm« (hours)

Figure la Acid formation, correlated to growth of Streptococcus cremoris 803 in the presence of 25 U lactoperoxidase

( _ * - - * - ) and in the absence of lacto-peroxidase (—• • — ) , tested in different media All media contained 2 mM thio-cyanate and 0 1 M glucose

Figure lb Acid formation, correlated to growth of Streptococcus cremoris 972 in the presence of 25 U lactoperoxidase ( _ ^ __:);_) and in the absence of lacto-peroxidase (—• • — ) , tested in different media All media contained 2 mM thio-cyanate and 0 1 M glucose

2 3 1 2 Inhibition of acid formation

The results of this canotron investigation (section 22 4 b) are given in table 2 The pH values given are the minimum measured a) when a 0.1 M glucose solution flowed on one side of the cell and b) the glucose solution was allowed to flow along the cells which had been previously conditioned by the passage for 10 minutes of the lactoperoxidase system (4U LPO-'ml, 2 m M S C N and 150 /<M H2O2) In both cases the pH minimum was reached in 10 to 15 minutes In this test the acid formation in all cases was almost the same as in the controls when one of the components of the lactoperoxidase s y s t e m w a s omitted Therefore to produce inhibition of acid formation it appeared t o be essential to have all three components of the lactoperoxidase system present.

(30)

Brain Heart Infusion broth

* Time (hours)

Tryptic Soy broth

.. Time (hours)

Lactobacillup broth, Merck

Figure 2 Acid formation, correlated to growth of Lactobacillus casei in the presence of 25 U lactoperoxidase ( - * - - * - ) and in the absence of lactoperoxidase (—• • — ) , tested in different media All media contained 2 mM thiocyanate and 0 1 M glucose

Brain Heart Infusion broth. Lactobacillus broth, Difco

Tryptic Soy broth. Lactobacillus broth, Merck

Figure 3 Acid formation, correlated to growth of Streptococcus mutans C 67-1 in the presence of 25 U lactoperoxidase ( - * - - * - ) and in the absence of lactoperoxidase (—• • — ) , tested in different media All media contained 2 mM thiocyanate and 0 1 M glucose

(31)

Minimum pH before application of the lactoperoxidase system 47 4 5 4.7 4 2 4.0 3 8 4.0 Minimum pH after applicat lactope system ion of the ^oxidase 4 9 4 5 4 3 6 0 65 61 62

Table 2 Effect of the lactoperoxidase system on the minimum pH of the test culture in the cariotron after rinsing with a 2% glucose solution

Test-organism Micrococcus aureus Proteus mirabilis Saccharomyces cerevisiae Streptococcus mutans C 67-1 Streptococcus mutans C 67 25 Streptococcus mutans OMZ 176 Lactobacillus casei

The growth inhibition of lactobacilli and streptococci by the lactoperoxidase system is dependent on the media in which the bacteria are grown Whilst in some media the inhibition of growth lasts for the length of the test, in others the lag-phase is only extended The results reported by various workers concerning the good inhibition of lactobacilli and the great differences within the strep-tococcal strains could not be reproduced, but their results are due to differences m the culture media used

Both Streptococcus mutans and sanguis and Streptococcus cremoris 803 generally reported to be resitant can be inhibited by the lactoperoxidase system under suitable conditions

2 3 2 Classification of bacteria forming lactic acid on the basis of their

sen-sitivity to the lactoperoxidase system

A s has been shown in section 2 3 1 the influence of the lactoperoxidase system on the micro-organisms is greatly affected by the substrate used

Therefore the classification medium was standardized to a weakly buffered solution with or without 0 05 M glucose

In these tests carried out at constant pH (2 2 4c) four different reaction patterns were found

a Micro-organisms inhibited in the presence of glucose when lactoperoxidase (4 U/ml) and thiocyanate ion (1 mM) were added

b Micro organisms inhibited in the presence of glucose when in addition to lactoperoxidase (5 U/ml) and thiocyanate (1 mM), hydrogenperoxide (150 fiM) w a s added

c Micro-organisms, inhibited by the lactoperoxidase system given under b, when this system was added to the cell suspension before the addition of glucose

d Micro-organisms inhibited as m c but only if the hydrogenperoxide was produced by glucoseoxidase

(32)

it

5 I

4 1

E

'\^r

h

i.

/

f/

T Y P E * 80 100 ' 2 0 MINUTES

Figure 4 Inhibition without addition of hydrogenperoxide in the presence of glucose Inhibition type a

It

* ,

100 120 MINUTES

Figure 5 Inhibition after addition of hydrogenperoxide in the presence of glucose Inhibition type b

6 5 4 3 2

i

^

O I E

t/

1 1

^%

1 -^z

» o « '

11 1/

/ TYPE C. 1 1 1 20 40 60 BO 100 120 MINUTES

Figure 6 Inhibition after addition of hydrogenperoxide in the presence of glucose Inhibition type c

Figures 4 7 Behaviour of various oral lactoperoxidase system de s E o S

i^l

\\\ 1/

100 120 MINUTES

Figure 7 Inhibition after addition of hydrogenperoxide and a hydrogenperoxide generating enzyme in the absence of glucose Inhibition type d

micro-organisms forming lactic acid towards the

This classification proved to be reproducable when the determining method was carefully standardized As hydrogenperoxide was likely to be formed in the media by the cells, both the cultures used and the time between addition of the

(33)

various components had to be carefully defined The cells were always harvested at the end of the logarithmic growth phase As the incubation time varied from strain to strain depending on the growth rate the initial dosage of 1 mM glucose after 17 minutes was a control on the acid forming properties of the cells used

In all cases approximately 0 1 m eq of acid was formed If cells were not inhibited by the addition of lactoperoxidase then 0 1 mM of hydrogenperoxide was added All cells which were still not inhibited, were tested according to procedure c and eventually procedure d

In all cases the criterion for inhibition was that the cells should form no acid for 30 minutes

In table 3 the reaction patterns of several micro-organisms are given

The oral strains tested were isolated from human plaque on sucrose plates according to the methods described by de Stoppelaar, 1971, using the clas-sification methods of Carlsson (1968) and Guggenheim (1968)

Strains 1, 2, 4, 7 and MW 25 (table 3) appeared to be Streptococcus mutans, whereas strains 9, 12, 15, 16, 21, 22 and 24 were identified as Streptococcus

mitis and strain 18 as Streptococcus sanguis

Strain MW 25, being hydrogenperoxide positive was classified by Hardie (London Hospital, Dental College) as being Streptococcus mutans, serotype d Table 3

Classification of some oral strains isolated from human dental plaque and their inhibition by the lactoperoxidase system

Number 1 2 4 7 9 12 15 16 18 21 24 MW25

1- ++ ++ ++ ++ ++ + +

Formation of H2O2 — Acid from sorbitol + Mannitol -I-5% sucrose medium ^) Soluble polysaccharides + + 4 % sodium chlonde -f-Raffmose -f Inulin -1-Aesculin — Ammonia from arginin —

Inhibition by LPO-system according to type b -f

+

')

+

—

+

b —

+

')

+ +

+

a —

+

-f-')

+ +

+

— b

— — — — — —— +

_ _ _ _ 2) 1)

— — — — -f-f — — -f +

+ -I- + + - I - — — —

— — — — — + + —

— - f - f - + + ± — -I- —

C O c c b b b

1) sticks to the wall

2) very viscous, but does not stick to the wall

In addition Streptococcus mutans C 67-1 was inhibited according to typing method b and Actinomyces viscosus to typing method d

(34)

2 3 3 Investigation of the three components of the lactoperoxidase system

2 3 3 1 Lactoperoxidase

The lactoperoxidase activity is often determined by a method using o-dianisidine This reagent is preferred due to its stability ( 2 2 7 ) thus fresh solutions do not have to be prepared for each determination The method has been used with good results in many reactions involving horseradish peroxidase However It appeared that with lactoperoxidase both iodide and thiocyanate ions

interfere when the o-dianisidme oi guaiacol method was used The results are given in the figures 8 and 9 If pyrogallol (EHiott, 1932) was used these ions did not interfere

o LU < 0 8 0 6 0 4 0 2 _ ^ T I M E [MINUTES]

Figure 8 The oxidation of guaiacol by lactoperox dase In the presence of halides measured as the increase in extinction at 470 nm as a function of time The concentrations of halides used were equivalent to those normally present in human saliva

— n n — 1 3 mM thiocyanate — O O — 1 «M iodide

—iV it— 15 mM chloride 50 '(M bromide 10 iiM fluoride or entirely free of halides

When carrying out the lactoperoxidase artivity determination it should be remembered that the enzyme has low stability in the presence of high

hydrogenperoxide concentrations This makes it impossible to use sufficiently high hydrogenperoxide concentrations to give a linear increase in the e ' t i n c t i o n The determination should be carried out in such a way as to give a minimum mactivation and the increase of extinction should be e/^tlapolated to time zero

(35)

TIME[ MINUTES]

Figure 9 As in figure 8 using o-dianisidine instead of guaiacol and measuring the extinction at 436 n m

Otherwise figures which are too low will be obtained

The average lactoperoxidase activity in the non-stimulated saliva of 15 people was found t o be 5 6 U / m l , with extreme values of 2 2 and 9 9 U / m l The standard deviation was 2 2 U/ml

A lactoperoxidase activity of 0 2 U''ml appeared to be sufficient to inhibit acid formation of most oral streptococci, if the hydrogenperoxide concentration was of the correct order

2 3 3 2 Thiocyanate

The average thiocyanate ion concentration in the non-stimulated saliva of 15 people was found to be 1 7 m M , with extreme values of 0 25 and 3 mM and a standard deviation of 0 7 The range is somewhat larger than found by Austin and Zeldow (1961)

It should be mentioned that the value of 0 25 mM was only found in one individual on one particular day This could be explained by the fact that changes in thiocyanate concentration can be due to dietary influences

The effect of thiocyanate concentration on the inhibition of acids, formed by a suspension of Streptococcus mutans, by the lactoperoxidase system was determined in test-tubes as described in section 2 2 4a

In the lactoperoxidase system the activity was kept constant at 5 U / m l , the concentration of hydrogenperoxide at 60 ^(M and the thiocyanate concentration

(36)

10,0 mM THIOCYANATE

Figure 10 pH's of cell supensions of Streptococcus mutans C 67-1 containing 5 U lactoperoxidase per ml and 60 ((M hydrogenperoxide, as a function of thiocyanate ion concentrations

Glucose (0 05 M) was added at time zero, pH was measured after 60 minutes (—[J D—) and 180 minutes (—it iV—)

10,0 mM THIOCYANATE

Figure 11 pH s of cell suspensions of Streptococcus mutans C 67-1 containing 5 U lactoperoxidase per ml and different concentrations of hydrogenperoxide of 15 ((M ( — • • — ) and 60 iiM (—r^ n—) respectively, as a function of thiocyanate ion concentrations

(37)

was varied from 0 to 10 mM The effect of thiocyanate was found to be rather constant within the limits found in saliva (figure 10)

As shown in figure 11 if a lower concentration of hydrogenperoxide was used (15 H M ) , the area of maximum inhibition moved to lower thiocyanate

con-centrations.

2 3 3 3. Hydrogenperoxide

When the effect of hydrogenperoxide concentration was examined using the plate method (section. 2.2.3.a), four distinct levels of hydrogenperoxide showed specific effects. At very low hydrogenperoxide concentrations inhibition only occurred if the micro-organism produced sufficient hydrogenperoxide itself. When the hydrogenperoxide concentration was about 0.1 m M there was definite growth inhibition.

In the range 10 to 100 mM no growth inhibition could be observed

A t still higher hydrogenperoxide concentrations the hydrogenperoxide itself had sufficient toxicity to prevent growth.

10 9 8 7 6

Is

E E c 4

i

S 3

s s s

E E E o o o S iQ " S E 3 E S _oa. o in 5 a.

8

s a. o CO >• H , 0 ,

Figure 12 Inhibition zones on plates as a function of the hydrogenperoxide concentration. Test organism Lactobacillus casei

Test conditions- Lactobacillus agar, Merck, 8 U lactoperoxidase in 0 1 ml solution, 10 mM thiocyanate and hydrogenperoxide as given.

(38)

6

-t .

E E E 4

.1

S 3 £ _c ~ 2 1 S E Z E S E S £

8 8 !2

a.

8

U1 3.

g

s

a.

s

a.

S

s a m S a. 01 H,0 2 U j

Figure 13 Inhibition zones on plates as a function of the hydrogenperoxide concentration Test organism Lactobacillus plantarum

Test conditions as given in figure 12

W h e n the test was repeated at increasing thiocyanate concentrations the area where no inhibition was found as a result of mactivation of the enzyme, moved to higher hydrogenperoxide concentrations Even with 20 mM thiocyanate ion there was no inhibition found with hydrogenperoxide concentrations of 15 mM The results are given in table 4

Table 4 Inhibition in mm on plates due to the lactoperoxidase system, at different concentrations of hydrogenperoxide and thiocyanate

Test organism Lactobacillus casei

Test conditions Lactobacillus agar, Merck Lactoperoxidase 8 U per hole containing 0 1 ml testsolution Molarity of testsolutions Thiocyanate 0,5 2,5 10 20 mM mM mM m M 2 E o 00

—

2 E i n

—

2 E 00

—

1 2 Hydrogenpe 2 E in

,_

- H V/2 272 2 E o V2 2 3 'oxide 2 in o V? 2 3 2 o CO VA 2 3 2 00

.

%

2 3 2 CO o ± 1 2 3 The different behaviour of Lactobacillus casei and arabinosus observed in these tests with low hydrogenperoxide concentrations resulted from hydrogen-peroxide formed by the micro organisms

(39)

s

60 -20

^ ^ WlNUrES

14-1 Cells harvested during lag-phase, 2 hours after inoculation

~9-r o • I

s

6 5 4 -" • ^ MINUTES • • ^ MINUTES ' « ^ MINUTES

14-2 Cells harvested 3 hours after start of loganthmic growth

60 ^ ^ MINUTES ' " ^ MINUTES

14-3 Cells harvested during stationary growth phase, 48 hours after inoculation Figure 14 Formation of hydrogenperoxide and acid by Streptococcus mutans C 67-1, in a 0 1 M glucose solution.

(40)

MINUTES

15-1 Cells harvested at the start of logarithmic growth

MINUTES O X Z 3 -30 60 ' W i ^ MINUTES

15-2 Cells harvested 4 hours after start of logarithmic growth

Figure 15 Formation of hydrogenperoxide and acid by Streptococcus mutans MW 25, in a 0 1 M glucose solution

Carlsson (1968) is the positive hydrogenperoxide reaction of sanguis if the micro-organism was cultured on a o-dianisidine plate (method section 2 2 7 ) . As the hydrogenperoxide formation is of importance f o r the correct functioning of the lactoperoxidase system, this aspect was further examined (method section 2 2 7 ) .

Although Streptococcus mutans C 67-1 gave a negative reaction on o-dianisidine plates it could form hydrogenperoxide in the tested media.

(41)

Strain M W 25 isolated from the oral flora in the course of the experimental work showed a stronger tendency to form hydrogenperoxide in the media, and even exhibited a positive effect on an o-dianisidine plate

The formation of hydrogenperoxide depended on the growth phase There was no correlation with the acid formation The formation of hydrogenperoxide was at the maximum with cells harvested during the stationary growth phase These results are given in figures 14 and 15

The effect of the hydrogenperoxide formed by the cells was investigated more fully in the following test Cells of Streptococcus mutans were harvested, washed and resuspended in a weakly buffered solution to which were added thiocyanate (1 mM) and glucose (0 1 M) A portion of this suspension v/as isolated immediately and then further portions every 10 minutes The pH and hydrogenperoxide content of each portion was measured and then 4 U ml lactoperoxidase was added After another 30 minutes the pH and hydrogen-peroxide content were measured once more

Results are given in table 5

Table 5 Effect of hydrogenperoxide formed by Streptococcus mutans on the inhibitory action of lactoperoxidase and thiocyanate

Addition of 4 U/ml lactoperoxidase at time 0 10 mm 20 mm 30 mm 60 mm pH at time of addition 6 5 6 1 5 3 4 6 4 25 pH 30 minutes later 4 75 4 4 5 5 2 4 6 4 25 pH of control without LPO 4 75 4 45 4 4 4 25 4 1 5 H2O2 concentration at time of addition 0 1 fiM 3 5 ((M 12 « M 18 ((M Inhibition

- -1--F -F

Inhibition occurred only after a certain hydrogenperoxide concentration was reached If lactoperoxidase was added before this concentrahon of hydrogen-peroxide was present no inhibition could be found After the lactoperoxidase was added no further hydrogenperoxide could be detected

Streptococcus mutans C 67-1 was inhibited only if the hydrogenperoxide con-centrations formed by the cells themselves exceeded 3 5 ^ M If inhibition was achieved with added hydrogenperoxide then higher concentrations were necessary

As seen from figure 16 under test conditions the inhibition at a hydrogen-peroxide concentration of 20 uM lasted for a period of 30 minutes

Doubling this concentration increased the period of inhibition to approximately 120 minutes At a hydrogenperoxide concentration of 50 /nM inhibition was

The effect of lactoperoxidase-thiocyanate-hydrogen peroxide on the metabolism of cariogenic micro-organisms in vitro and in the oral cavity

THE EFFECT OF

LACTOPEROXIDASE-THIOCYANATE-HYDROGEN PEROXIDE ON THE

METABOLISM OF CARIOGENIC

MICRO-ORGANISMS IN VITRO AND

IN THE ORAL CAVITY

H. HOOGENDOORN

THE EFFECT OF

LACTOPEROXIDASE-THIOCYANATE-HYDROGEN PEROXIDE ON THE

METABOLISM OF CARIOGENIC

MICRO-ORGANISMS IN VITRO AND

IN THE ORAL CAVITY

PROEFSCHRIFT

/B/0 ^/OVO

Dankwoord

Contents

Chapter 1

INTRODUCTION AND SURVEY

OF LITERATURE

1.1. Development and prevention of carious lesions

1 2 The caries preventive activity of saliva

1.3. Lactoperoxidase as a bacteriostatic factor in saliva

i

Chapter 2

EXPERIMENTAL

2.1. Aim of the investigations

2 2 Materials and methods

2.3. Specific experiments and results

it

4 1

'\^r

h

i.

/

f/

It

i

^

t/

^%

1 -^z

11 1/

i^l

\\\ 1/

1- ++ ++ ++ ++ ++ + +

+

+

+

+

+

+

+

+

+

+ +

+

+

+

+

+

+

+ +

+

+

+

+

— — — — — —— +

— — — — -f-f — — -f +

+ -I- + + - I - — — —

— — — — — + + —

— - f - f - + + ± — -I- —

Is

i

s s s

8

-t .

.1

8 8 !2

8

g