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Objective: To investigate interactions between hyaluronic acid (HA), lysozyme, and the glucose oxidase-mediated lactoperoxidase (GO-LPO) system in enzymatic and candidacidal activities., Design: The influences of HA (0.5, 1.0, and 2.0mg/mL) and lysozyme (30μg/mL hen egg white lysozyme) on the enzymatic activity of GO-LPO system (25μg/mL bovine LPO, 1mM KSCN, 10units/mL GO, and 30μg/mL glucose) were determined by measuring oxidized o-dianisidine production. The influence of the GO-LPO system on lysozyme activity was determined by measuring the turbidity of a Micrococcus lysodeikticus suspension. The effects of interactions between HA, lysozyme, the GO-LPO system on candidacidal activity were examined by pre-incubating various combinations of components. Candidacidal activity was determined by comparing the numbers of colony forming units using Candida albicans ATCC strains 10231, 18804, and 11006., Results: HA inhibited the enzymatic activity of the GO-LPO system in a dose-dependent manner. HA inhibited the candidacidal activities of the GO-LPO system. However, the inhibitory activity of HA was not significantly different according to concentration of HA. The GO-LPO system enhanced the enzymatic activity of lysozyme, though lysozyme did not affect the enzymatic activity of the GO-LPO system. The candidacidal activities of the GO-LPO system and lysozyme were not additive., Conclusions: HA inhibited the enzymatic and candidacidal activity of the GO-LPO system. The GO-LPO system enhanced the enzymatic activity of lysozyme, but the candidacidal activities were not additive., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study was to investigate the candidacidal activity of the glucose oxidase-mediated lactoperoxidase system at various levels of glucose and glucose oxidase., Design: Candida albicans ATCC strains 18804, 10231, 11006, bovine lactoperoxidase (25 and 50 μg/mL), and KSCN (1 mM) were used. Different levels of glucose oxidase (1, 5, 10, and 20 units/mL) and glucose (0.03, 0.3, and 3.0 mg/mL) were added to complete the system. The candidacidal activity of the system was examined by preincubating its components for 0-60 min, and then with C. albicans. Candidacidal activity was determined by comparing the numbers of CFU and calculating the percent loss of cell viability., Results: The system displayed 13.9-27.4% (without preincubation) to 28.6-34.3% (preincubation for 60 min) loss of viability at 25 μg/mL of bovine lactoperoxidase, 10 units/mL of glucose oxidase, and 0.03 mg/mL of glucose; similar results were obtained with 20 units/mL of glucose oxidase or 0.06 mg/mL of glucose. The candidacidal activity of the system increased markedly as the glucose concentration increased. The candidacidal activity displayed 87.2% (without preincubation) to 100.0% (preincubation for 60 min) at 3.0 mg/mL of glucose. At 3.0 mg/mL of glucose, the system containing 1 or 5 units of glucose oxidase also showed significant levels of candidacidal activity., Conclusions: The candidacidal activity of the glucose oxidase-mediated lactoperoxidase system at physiological concentrations of salivary glucose was moderate, but was greatly elevated with increases of glucose level., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: As shown in the quantitative suspension test adding lactoperoxidase to a thiocyanate (SCN(-)) hydrogen peroxide (H(2)O(2)) combination over the physiological saliva level has significant positive antimicrobial effects to a level of totally killing Streptococcus mutans, Streptococcus sanguinis, and Candida albicans. The aim of this study was to evaluate this positive effect under human saliva loading., Methods: The bactericidal and fungicidal effect of lactoperoxidase was evaluated in a quantitative suspension test by using two test mixtures of a 2.0% thiocyanate and 1.2% hydrogen peroxide solution, one without (Group A) and one with (Group B) lactoperoxidase under saliva loading. Following the quantitative suspension tests (EN-13727/EN-13624), the growth of surviving bacteria and fungi in a nutrient broth was measured. The exposure times were restricted to 1, 3, 5, and 15 min. All statistical analyses were carried out with SPSS 11.5., Results: In the quantitative suspension test, the combination of thiocyanate and hydrogen peroxide showed relatively low antimicrobial effectiveness on S. mutans, S. sanguinis, and C. albicans in the presence of human saliva at measured time points in comparison to the mixture with lactoperoxidase, which showed a high bactericidal activity within 15 min (S. mutans and S. sanguinis) and fungicidal activity within 3 min (C. albicans)., Conclusion: The antimicrobial effectiveness of the tested thiocyanate hydrogen peroxide combination was increased significantly by adding lactoperoxidase in the quantitative suspension test under human saliva loading., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effects of peroxidase or the peroxidase system on the enzymatic and candidacidal activities of lysozyme., Design: The effects of peroxidase on lysozyme were examined by incubating hen egg-white lysozyme (HEWL) with bovine lactoperoxidase (bLPO). The influence of the peroxidase system on lysozyme was examined by the subsequent addition of potassium thiocyanate and hydrogen peroxide. Lysozyme activity was determined by the turbidity measurement of a Micrococcus lysodeikticus substrate suspension. Candidacidal activity was determined by comparing the colony forming units of Candida albicans ATCC 10231, ATCC 18804, and ATCC 11006. The Wilcoxon signed rank test was used to analyze the effects of variables., Results: bLPO at physiological concentrations enhanced the enzymatic activity of HEWL and its effect was dependent on bLPO concentration. The enhancement of enzymatic activity of HEWL by bLPO was affected by pH and ionic strength. The addition of potassium thiocyanate and hydrogen peroxide did not lead to an additional enhancement of the enzymatic activity of HEWL, as compared with bLPO alone. HEWL displayed candidacidal activity in all 3 strains of C. ablicans. The addition of bLPO alone did not affect the candidacidal activity of HEWL, but the bLPO system enhanced candidacidal activity of HEWL in all 3 strains of C. ablicans., Conclusions: bLPO enhanced the enzymatic activity of HEWL, but the bLPO system did not show additional enhancement of the enzymatic activity of HEWL. The addition of bLPO did not affect the candidacidal activity of HEWL, but the bLPO system did enhance the candidacidal activity of HEWL., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Aim: Mouth rinses containing enzymes are designed for patients suffering from xerostomia. The objective of the present in situ study was to investigate the efficacy of these rinses for targeted accumulation and immobilisation of protective enzymes in the acquired pellicle., Methods: A number of six healthy subjects carried bovine enamel slabs fixed on individual upper jaw splints for pellicle formation in situ. After 1 min, they rinsed with biotène or BioXtra for 10 min, respectively. Enzyme activities of lysozyme, peroxidase and glucoseoxidase in the in situ pellicle and in the saliva were assayed before as well as 0, 20 and 40 min after the rinses. The assays for the respective enzyme activities were based on fluorogenic substrates. Separate experiments were performed for the different enzymes and mouth rinses, respectively. Statistical evaluation was carried out with the Kruskal-Wallis test., Results: None of the investigated rinses had any significant impact on the activities of lysozyme, peroxidase and glucoseoxidase detectable in the in situ pellicle or in the saliva (Kruskal-Wallis test, p>0.05). Despite the fact that both products should contain lactoperoxidase activity according to manufacturers' instructions, no peroxidase activity was measurable in the pure mouth rinses., Conclusion: With the tested enzymatic mouth rinses targeted accumulation and immobilisation of protective enzymes in the in situ pellicle did not seem possible., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Teeth were inoculated with either the organisms separately or with a freshly-prepared mixture of both. The apparatus was swept with 5 per cent (v/v) CO2 in either air or N2, and incubated for 90 h. A nutrient supplement containing 1 per cent (w/v) glucose was supplied for 1 h in every 6 h. Both organisms achieved similar numbers when grown aerobically in pure culture, yet in mixed culture there was pronounced inhibition of BHT (p less than 0.001). When the synthetic saliva was supplemented with catalase the strain BHT count in mixed culture was much higher (p less than 0.001). It was concluded, therefore, that the strain LPA-1 produced inhibitory levels of hydrogen peroxide (H2O2) on the tooth surface under aerobic conditions. This was supported by finding that a lower viable count of LPA-1 in pure culture was attained when lactoperoxidase (LPO) was included in the saliva (p less than 0.005), as all components of the LPO-SCN-H2O2 system were presumably present. With the N2-CO2 mixture, conditions were not strictly anaerobic and both catalase and LPO increased all viable counts. Under these conditions, therefore, when H2O2 was limiting, LPO protected bacteria against its bactericidal effect.

The hypothiocyanite ion (OSCN-) is the principal oxidation product of the salivary peroxidase-thiocyanate (SCN-)-hydrogen peroxide antimicrobial system. Supplementation of human saliva in vitro and in vivo with low amounts (less than 1.0 mM) of hydrogen peroxide increase the concentration of salivary OSCN- (in vivo up to 0.3 mM). Elevated concentrations of OSCN- are strongly antimicrobial and may therefore be protective against dental caries. However, as OSCN- is a highly-reactive oxidizing agent, its possible toxic effect on human cells was studied using gingival fibroblasts as target cells. Concentrations of OSCN- (up to 300 microM) had no effect on [3H]-thymidine incorporation into the cells. However, fibroblasts were sensitive to peroxide so that 100 microM of H2O2 caused over 80 per cent reduction in [3H]-thymidine incorporation. The toxicity of H2O2 could be entirely prevented by adding lactoperoxidase and SCN- to the cell culture before the addition of peroxide. Thus, conversion of toxic H2O2 to non-toxic OSCN- in fibroblast culture by lactoperoxidase and SCN- suggests a dual role for the salivary peroxidase system: protection of human cells from H2O2 toxicity and antimicrobial action against oral pathogens. Furthermore, the elevated concentrations of OSCN- which produce inhibition of bacterial metabolism did not damage human cells.