Comparative Analysis of the Antiviral Activity of Camel, Bovine, and Human Lactoperoxidases Against Herpes Simplex Virus Type 1.

Lactoperoxidase is a milk hemoprotein that acts as a non-immunoglobulin protective protein and shows strong antimicrobial activity. Bovine milk contains about 15 and 7 times higher levels of lactoperoxidase than human colustrum and camel milk, respectively. Human, bovine, and camel lactoperoxidases (hLPO, bLPO, and cLPO, respectively) were purified as homogeneous samples with specific activities of 4.2, 61.3, and 8.7 u/mg, respectively. The optimal working pH was 7.5 (hLPO and bLPO) and 6.5 (cLPO), whereas the optimal working temperature for these proteins was 40 °C. The K m of hLPO, cLPO, and bLPO were 17, 16, and 19 mM, and their corresponding V max values were 2, 1.7, and 2.7 µmol/min ml. However, in the presence of H2O2, the K m values were 11 mM for hLPO and cLPO and 20 mM for bLPO, while the corresponding V max values were 1.17 for hLPO and 1.4 µmol/min ml for cLPO and bLPO. All three proteins were able to inhibit the herpes simplex virus type 1 (HSV-1) in Vero cell line model. The relative antiviral activities were proportional to the protein concentrations. The highest anti-HSV-1 activity was exhibited by bLPO that inhibited the HSV particles at a concentration of 0.5 mg/ml with the relative activity of 100%.

Milk-derived proteins and peptides in clinical trials.

Clinical trials are reviewed, involving proteins and peptides derived from milk (predominantly bovine), with the exception of lactoferrin, which will be the subject of another article. The most explored milk fraction is α-lactalbumin (LA), which is often applied with glycomacropeptide (GMP) - a casein degradation product. These milk constituents are used in health-promoting infant and adult formulae as well as in a modified form (HAMLET) to treat cancer. Lactoperoxidase (LCP) is used as an additive to mouth hygiene products and as a salivary substitute. Casein derivatives are applied, in addition, in the dry mouth syndrome. On the other hand, casein hydrolysates, containing active tripeptides, found application in hypertension and in type 2 diabetes. Lysozyme is routinely used for food conservation and in pharmaceutical products. It was successfully used in premature infants with concomitant diseases to improve health parameters. When used as prophylaxis in patients with scheduled surgery, it significantly reduced the incidence of hepatitis resulting from blood transfusion. Lysozyme was also used in infected children as an antimicrobial agent showing synergistic effects in combination with different antibiotics. Proline-rich polypeptide (PRP) was introduced to therapy of Alzheimer's disease patients. The therapeutic value of PRP was proved in several clinical trials and supported by studies on its mechanism of action. Concentrated immunoglobulin preparations from colostrum and milk of hyperimmunized cows showed efficacy in prevention of infections by bacteria, viruses and protozoa. A nutrition formula with milk-derived TGF-ß2 (Modulen IBD®) found application in treatment of pediatric Crohn's disease. In conclusion, the preparations containing milk-derived products are safe and effective measures in prevention and treatment of infections as well as autoimmune and neoplastic diseases.

Antibacterial activity of the lactoperoxidase system combined with edible Laminaria hot-water extract as a source of halide ions.

Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI(-) concentration in the assay mixture after ultrafiltration.

Effect of lactoperoxidase system containing toothpaste on cariogenic bacteria in children with early childhood caries.

BACKGROUND AND OBJECTIVES: Lactoperoxidase system contains Lactoperoxidase, Hydrogen peroxide and Thiocyanate ions, which have inhibitory action against cariogenic oral microflora. The present study was undertaken to assess the effect of lactoperoxidase system containing toothpaste on cariogenic microflora in children with early childhood caries. METHODS: Study group included 30 children with Early Childhood Caries. 15 were considered as test group who used the test product Biotene toothpaste and other 15 as control group who used Colgate Active as control product. Salivary samples were analyzed for mutans streptococci (MS) and Lactobacilli, and for the levels of Thiocyanate ions. RESULTS: Showed significant increase in the levels of Thiocyanate ion in saliva during experimental period. Compared to the control group test group showed significant increase in the levels of thiocyanate ions during experimental and washout period, whereas the number of colonies of MS and Lactobacilli were significantly reduced in test group during experimental period. CONCLUSION: The levels of thiocyanate ions can be increased in vivo by supplementing the saliva with natural enzymes like lactoperoxidase. This increased concentration of thiocyanate will reduce the number of cariogenic microflora in children with Early Childhood Caries.

Challenge testing the lactoperoxidase system against a range of bacteria using different activation agents.

Lactoperoxidase (LP) exerts antimicrobial effects in combination with H(2)O(2) and either thiocyanate (SCN(-)) or a halide (e.g., I(-)). Garlic extract in the presence of ethanol has also been used to activate the LP system. This study aimed to determine the effects of 3 LP activation systems (LP+SCN(-)+H(2)O(2); LP+I(-)+H(2)O(2); LP + garlic extract + ethanol) on the growth and activity of 3 test organisms (Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus). Sterilized milk was used as the reaction medium, and the growth pattern of the organisms and a range of keeping quality (KQ) indicators (pH, titratable acidity, ethanol stability, clot on boiling) were monitored during storage at the respective optimum growth temperature for each organism. The LP+I(-)+ H(2)O(2) system reduced bacterial counts below the detection limit shortly after treatment for all 3 organisms, and no bacteria could be detected for the duration of the experiment (35 to 55 h). The KQ data confirmed that the milk remained unspoiled at the end of the experiments. The LP + garlic extract + ethanol system, on the other hand, had no effect on the growth or KQ with P. aeruginosa, but showed a small retardation of growth of the other 2 organisms, accompanied by small increases (5 to 10 h) in KQ. The effects of the LP+SCN(-)+H(2)O(2) system were intermediate between those of the other 2 systems and differed between organisms. With P. aeruginosa, the system exerted total inhibition within 10 h of incubation, but the bacteria regained viability after a further 5 h, following a logarithmic growth curve. This was reflected in the KQ indicators, which implied an extension of 15 h. With the other 2 bacterial species, LP+SCN(-)+H(2)O(2) exerted an obvious inhibitory effect, giving a lag phase in the growth curve of 5 to 10 h and KQ extension of 10 to 15 h. When used in combination, I(-) and SCN(-) displayed negative synergy.

Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactoferrin in xerostomia: efficacy and safety.

Innate human salivary defence proteins, lysozyme, lactoferrin and peroxidase, are known to exert a wide antimicrobial activity against a number of bacterial, viral and fungal pathogens in vitro. Therefore, these proteins, alone or in combinations, have been incorporated as preservatives in foods and pharmaceuticals as well as in oral health care products to restore salivas' own antimicrobial capacity in patients with dry mouth. These antimicrobials used in oral health care products, such as dentifrices, mouth-rinses, moisturizing gels and chewing gums, have been purified from bovine colostrum. In this review I critically evaluate the clinical efficacy and safety of this kind of preventive approach against various oral diseases and symptoms.

Effects of three different infant dentifrices on biofilms and oral microorganisms.

The purpose of this work was to evaluate the effects of infant dentifrices: A--with lactoperoxidase, glucose oxidase and lactoferrin; B--with 1100 ppm of NaF and sodium lauryl sulfate; C--with extract of calendula. The dentifrices were test on biofilms formed in vitro from saliva and dental plaque of infants, using reference strains A. viscosus (ATCC 43146); C. albicans (ATCC 51501); L. casei (ATCC 4646); S. mitis (ATCC 49456); S. mutans (ATCC 25175); S. oralis (ATCC 35037); S. sanguis (ATCC 10586); S. sobrinus (ATCC 27609) and isolated clinically microorganisms C. albicans, S. mitis, S. mutans, S. oralis, S. sanguis, S. sobrinus and Lactobacillus sp. Twenty infants were chosen, who were beginning treatment at the Infants Clinic of the Pediatric Dentistry Department, Federal University of Rio de Janeiro. A pool of unstimulated saliva and a pool of dental plaque were collected from which biofilms were produced. Supernatants from each dentifrice were prepared and concentrated and diluted solutions of the dentifrices and a control sterile diluent were tested against the biofilms produced, for 1 and 3 minutes, and against the microorganisms. The results were statistically analyzed by the ANOVA and Tukey Test. After the exposure of the biofilms produced both from saliva and from dental plaque, to the dentifrice B concentrated and 1/2, for 1 and 3 minutes, the viable microorganisms count (CFU/ml), compared to the controls, was significantly reduced (p < 0.05). However, exposure to the dentifrices A and C concentrated and dentifrice B 1/4 and 1/8, for 1 and 3 minutes, was not significantly lethal to the biofilms. The dentifrices A and C, either concentrated or diluted (1/2 to 1/128) and the dentifrice B in the dilutions 1/16 to 1/128 did not have an antimicrobial effect on any microorganism evaluated. For all the microorganisms evaluated, the dentifrice B concentrated and in the 1/2 dilution showed a significant antimicrobial effect, when compared with the control (p < 0.05).

Inhibition of Candida albicans by the Peroxidase/SCN-/H2O2 system.

Effects of the salivary peroxidase (SPO) system on the growth, glucose uptake and metabolic activities of oral bacteria are well documented but the effects on oral fungi are virtually unknown. Therefore, the viability of Candida albicans (ATCC 28366) exposed to the peroxidase/SCN-/H2O2 system was studied in sterilized saliva, in phosphate-buffered saline (PBS) and in potassium chloride. The growth of C. albicans in glucose-supplemented saliva was faster at pH 5.5 than at pH 7. The addition of the complete SPO (or lactoperoxidase) system to either sterilized saliva, KCl (50 microM) or PBS at pH 5.5 inhibited dose-dependently the viability of C. albicans in KCl, but no inhibition was found in PBS or saliva. Maximal inhibition was achieved in 2 h and with > 320 microM of peroxidase-generated HOSCN/OSCN-. However, physiological salivary concentrations of phosphate (> or = 1.0 mM) and PBS blocked the antifungal effect of HOSCN/OSCN-. The relative proportions of SCN- and H2O2 were critical to the antifungal effects. With 0.2 mM KSCN, a complete loss of viability was achieved, though the HOSCN/OSCN- concentrations did not exceed 100 microM. It is concluded that C. albicans is sensitive to HOSCN/OSCN- but salivary concentrations of phosphate block the antifungal effect of the peroxidase systems.

The protective effect of peroxidase and thiocyanate against hydrogen peroxide toxicity assessed by the uptake of [3H]-thymidine by human gingival fibroblasts cultured in vitro.

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.

Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans.

The antimicrobial effect of the lactoperoxidase (LPO) system (enzyme with the thiocyanate ion and hydrogen peroxide) on Streptococcus mutans NCTC 10449 (serotype c) was significantly enhanced when the system was combined with secretory IgA. Similar enhancement was observed with LPO-myeloma IgA1 or IgA2 combinations. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between LPO and immunoglobulin. However, neither human polyclonal nor myeloma IgG or IgM nor rabbit IgG enhanced the antibacterial activity of the LPO system. None of the immunoglobulins, when added alone, produced antimicrobial effects. LPO was shown to bind to colostral secretory IgA, myeloma IgA1, IgA2, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM. This binding had a stabilizing effect on the enzyme activity. Our results suggest that IgA significantly enhances the antibacterial efficiency of one of the innate immune factors--the LPO system.