Epithelial chemerin-CMKLR1 signaling restricts microbiota-driven colonic neutrophilia and tumorigenesis by up-regulating lactoperoxidase.

Intestinal barrier immunity is essential for controlling gut microbiota without eliciting harmful immune responses, while its defect contributes to the breakdown of intestinal homeostasis and colitis development. Chemerin, which is abundantly expressed in barrier tissues, has been demonstrated to regulate tissue inflammation via CMKLR1, its functional receptor. Several studies have reported the association between increased expression of chemerin-CMKLR1 and disease severity and immunotherapy resistance in inflammatory bowel disease (IBD) patients. However, the pathophysiological role of endogenous chemerin-CMKLR1 signaling in intestinal homeostasis remains elusive. We herein demonstrated that deficiency of chemerin or intestinal epithelial cell (IEC)-specific CMKLR1 conferred high susceptibility to microbiota-driven neutrophilic colon inflammation and subsequent tumorigenesis in mice following epithelial injury. Unexpectedly, we found that lack of chemerin-CMKLR1 signaling specifically reduced expression of lactoperoxidase (LPO), a peroxidase that is predominantly expressed in colonic ECs and utilizes H2O2 to oxidize thiocyanates to the antibiotic compound, thereby leading to the outgrowth and mucosal invasion of gram-negative bacteria and dysregulated CXCL1/2-mediated neutrophilia. Importantly, decreased LPO expression was causally linked to aggravated microbiota-driven colitis and associated tumorigenesis, as LPO supplementation could completely rescue such phenotypes in mice deficient in epithelial chemerin-CMKLR1 signaling. Moreover, epithelial chemerin-CMKLR1 signaling is necessary for early host defense against bacterial infection in an LPO-dependent manner. Collectively, our study reveals that the chemerin-CMKLR1/LPO axis represents an unrecognized immune mechanism that potentiates epithelial antimicrobial defense and restricts harmful colonic neutrophilia and suggests that LPO supplementation may be beneficial for microbiota dysbiosis in IBD patients with a defective innate antimicrobial mechanism.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Oxidation of anti-thyroid drugs and their selenium analogs by ABTS radical cation.

Lactoperoxidase was previously used as a model enzyme to test the inhibitory activity of selenium analogs of anti-thyroid drugs with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. Peroxidases oxidize ABTS to a metastable radical ABTS•+, which is readily reduced by many antioxidants, including thiol-containing compounds, and it has been used for decades to measure antioxidant activity in biological samples. We showed that anti-thyroid drugs 6-n-propyl-2-thiouracil, methimazole, and selenium analogs of methimazole also reduced it rapidly. This reaction may explain the anti-thyroid action of many other compounds, particularly natural antioxidants, which may reduce the oxidized form of iodine and/or tyrosyl radicals generated by thyroid peroxidase thus decreasing the production of thyroid hormones. However, influence of selenium analogs of methimazole on the rate of hydrogen peroxide consumption during oxidation of ABTS by lactoperoxidase was moderate. Direct hydrogen peroxide reduction, proposed before as their mechanism of action, cannot therefore account for the observed inhibitory effects. 1-Methylimidazole-2-selone and its diselenide were oxidized by ABTS•+ to relatively stable seleninic acid, which decomposed slowly to selenite and 1-methylimidazole. In contrast, oxidation of 1,3-dimethylimidazole-2-selone gave selenite and 1,3-dimethylimidazolium cation. Accumulation of the corresponding seleninic acid was not observed.

Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies.

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC50 ) value and an inhibition constant (Ki ) values of methyl benzoates were determined. These compounds inhibited LPO with Ki values ranging from 0.033±0.004 to 1540.011±460.020 µM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (Ki =0.033±0.004 µM). The most potent inhibitor (1 a) showed with a docking score of -3.36 kcal/mol and an MM-GBSA value of -25.05 kcal/mol, of these methyl benzoate derivatives (1 a-16 a) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).

Safety Assessment of the Modified Lactoperoxidase System-In Vitro Studies on Human Gingival Fibroblasts.

One strategy in caries prevention is to inhibit the formation of cariogenic biofilms. Attempts are being made to develop oral hygiene products enriched with various antimicrobial agents. One of them is lactoperoxidase-an enzyme that can oxidise (pseudo)halide ions to reactive products with antimicrobial activity. Currently, commercially available products utilise thiocyanate as a substrate; however, several alternatives that are oxidised to products with greater antimicrobial potential have been found. In this study, toxicity against human gingival fibroblasts of the lactoperoxidase system was evaluated using four different (pseudo)halide substrate systems-thiocyanate, iodide, selenocyanate, and a mixture of thiocyanate and iodide. For this purpose, cells were treated with the systems and then apoptosis, cell cycle, intracellular glutathione concentration, and mitochondrial superoxide production were assessed. The results showed that each system, after generating 250 µM of the product, inhibited cell divisions, increased apoptosis, and increased the percentage of dead cells. It was concluded that the mechanism of the observed phenomena was not related to increased superoxide production or the depletion of glutathione concentration. These findings emphasised the need for the further in vitro and in vivo toxicity investigation of the modified lactoperoxidase system to assess its safety and the possibility of use in oral hygiene products.

Modified Lactoperoxidase System as a Promising Anticaries Agent: In Vitro Studies on Streptococcus mutans Biofilms.

The lactoperoxidase (LPO) system shows promise in the prevention of dental caries, a common chronic disease. This system has antimicrobial properties and is part of the non-specific antimicrobial immune system. Understanding the efficacy of the LPO system in the fight against biofilms could provide information on alternative strategies for the prevention and treatment of caries. In this study, the enzymatic system was modified using four different (pseudo)halide substrates (thiocyanate, thiocyanate-iodide mixture, selenocyanate, and iodide). The study evaluated the metabolic effects of applying such modifications to Streptococcus mutans; in particular: (1) biofilm formation, (2) synthesis of insoluble polysaccharides, (3) lactate synthesis, (4) glucose and sucrose consumption, (5) intracellular NAD+ and NADH concentrations, and (6) transmembrane glucose transport efficiency (PTS activity). The results showed that the LPO-iodide system had the strongest inhibitory effect on biofilm growth and lactate synthesis (complete inhibition). This was associated with an increase in the NAD+/NADH ratio and an inhibition of glucose PTS activity. The LPO-selenocyanate system showed a moderate inhibitory effect on biofilm biomass growth and lactate synthesis. The other systems showed relatively small inhibition of lactate synthesis and glucose PTS but no effect on the growth of biofilm biomass. This study provides a basis for further research on the use of alternative substrates with the LPO system, particularly the LPO-iodide system, in the prevention and control of biofilm-related diseases.

Comments on the Discussion Forum: Oromucosal immunomodulation as clinical spectrum mitigating factor in SARS-CoV-2 infection.

The mammalian lactoperoxidase system, consisting of lactoperoxidase and the H2 O2 -producing enzyme duox, is our first line of defence against airborne microbes. This system catalyses the production of hypoiodite and hypoiodous acid in the presence of sufficient iodine. These products are highly efficient at destroying the H1N1 virus and the respiratory syncytial virus (RSV). Japan has not been affected as much as other nations during the COVID-19 pandemic (death rate about 10% of the United States), and we think this is due to a diet high in iodine. With this in mind, we suggest four actions to prevent SARS-CoV-2 infections. First, health professionals should study the preventative effect of increasing iodine in the diets of the aged, institutionalized, diabetics andsmokers. Second, the recommended daily intake (RDI) for iodine should be significantly increased, to at least double, the current RDI. Governments should encourage the use and distribution of cheap iodized salts, kelp and seaweed. Third, more research should be done around the physiology and the protective effects of the lactoperoxidase system. Finally, the degradation products of the SARS-CoV-2 viral particle by hypoiodite and hypoiodous acid should be characterized; portions of the damaged particle are likely to elicit stronger immunity and better vaccines.

Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

In vitro effects of standard antioxidants on lactoperoxidase enzyme-A molecular docking approach.

Lactoperoxidase (LPO), an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products. This study aimed to determine the in vitro inhibition effects of Trolox, α-tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate, which are commonly used as antioxidants in food and pharmaceutical products. For this purpose, LPO was first purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography. Also, some inhibition parameters, including half-maximal inhibitory concentration (IC50 ), Ki values, and inhibition types, were calculated for each antioxidant molecule. The IC50 values of these molecules, which exhibited competitive inhibition, varied between 377.7 and 3397.8 nM. Molecular docking studies were also performed for all compounds. According to the binding scores, α-tocopherol was shown to exhibit the most effective inhibitor property (IC50 : 377.7 nM and Ki : 635.8 ± 16.8 nM) among the standard antioxidants used in this study. Inhibiting the LPO activity by standard antioxidants results in the weakening of the immune system during lactation, which is important for metabolism.

Structure elaboration of isoniazid: synthesis, in silico molecular docking and antimycobacterial activity of isoniazid-pyrimidine conjugates.

Designing small molecule-based new drug candidates through structure modulation of the existing drugs has drawn considerable attention in view of inevitable emergence of resistance. A new series of isoniazid-pyrimidine conjugates were synthesized in good yields and evaluated for antitubercular activity against the H37Rv strain of Mycobacterium tuberculosis using the microplate Alamar Blue assay. Structure-anti-TB relationship profile revealed that conjugates 8a and 8c bearing a phenyl group at C-6 of pyrimidine scaffold were most active (MIC99 10 µM) and least cytotoxic members of the series. In silico docking of 8a in the active site of bovine lactoperoxidase as well as a cytochrome C peroxidase mutant N184R Y36A revealed favorable interactions similar to the heme enzyme catalase peroxidase (KatG) that activates isoniazid. This investigation suggests a rationale for further work on this promising series of antitubercular agents.

Immobilization of lactoperoxidase on Fe3O4 magnetic nanoparticles with improved stability.

OBJECTIVE: The study aimed to develop a facile and effectual method to increase the stability of lactoperoxidase (LPO) by using its immobilization on Fe3O4 magnetic nanoparticles (Fe3O4 MNPs). RESULTS: The successful immobilization of LPO on Fe3O4 MNPs was confirmed by using Fourier transform infrared spectroscopy (FT-IR) and field emission scanning electron microscopy (FE-SEM). The Km values of free LPO and LPO immobilized on Fe3O4 were 53.19, 72.46 mM and their Vmax values were 0.629, 0.576 µmol/mL min respectively. The overall results indicated that the stability of the immobilized LPO was significantly improved compared to free LPO. The LPO immobilized on Fe3O4 (LPO- Fe3O4) retained 28% of the initial activity within 30 days at 25 °C whereas the free enzyme lost its activity after 7 days at the same temperature. Moreover, evaluation of the thermal stability of LPO at 75 °C determined the conservation of 19% of the initial activity of LPO in the LPO- Fe3O4 sample after 60 min whereas the free enzyme lost its activity after 5 min. CONCLUSIONS: According to the present results, Fe3O4 magnetic nanoparticles are suitable for the immobilization of LPO.

Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.

The lactoperoxidase system (LS), an antimicrobial system naturally present in milk that is activated by H2O2, has been used to inhibit microbial outgrowth in raw milk in areas where refrigeration is not viable. This study evaluated lactose oxidase (LO) as a novel activator of the LS. Lactose oxidase oxidizes lactose and produces H2O2 needed for the activation of the LS. The antimicrobial effect of different concentrations of LO with and without components of the LS, thiocyanate (TCN) and lactoperoxidase (LP), was evaluated in model systems and then applied in pasteurized milk and raw milk. In general, an increase in LO caused greater reductions of Pseudomonas fragi in the model systems and treatments were more effective at 6°C than at 21°C. At 6°C, the LO solution at 0.12 and 1.2 g/L showed significantly higher microbial reduction than the control when both added alone and combined with LS components. At 21°C, treatments with 1.2 g/L of LO solution achieved a reduction of >2.93 log cfu/mL in 24 h, but at lower levels there was not a significant reduction from the control. Higher concentrations of TCN led to a greater P. fragi reduction at both temperatures when LO was added alone but not when combined with LP. In pasteurized milk, the LO solution at 0.12 g/L caused a reduction of approximately 1.4 log of P. fragi within 24 h when added alone and a reduction of approximately 2.7 log when combined with LP and TCN. Bacterial counts remained at significantly lower levels than the control during storage, and the TCN-supplemented milk exhibited an approximately 6-log difference from the control by d 7. In raw milk, the total bacterial growth curve showed a longer lag phase when the LS was activated by LO (11.3 ± 1.4 h) compared with the control (4.0 ± 1.0 h), but it was not different from the recommended method (9.4 ± 1.0 h). However, the total bacterial count after 24 h for the sample treated with LO and TCN (5.3 log cfu/mL) was significantly lower compared with the control (7.2 log cfu/mL) and the recommended method (6.1 log cfu/mL). Results from this study suggest that LO is an alternative source of H2O2 that enhances the microbial inhibition achieved by the LS. Lactose oxidase could be used to develop enzyme-based preservation technologies for applications where cold chain access is limited. This enzymatic approach to improving the shelf life of dairy products also represents a novel option for clean label spoilage control.

The Significance of Lactoperoxidase System in Oral Health: Application and Efficacy in Oral Hygiene Products.

Lactoperoxidase (LPO) present in saliva are an important element of the nonspecific immune response involved in maintaining oral health. The main role of this enzyme is to oxidize salivary thiocyanate ions (SCN-) in the presence of hydrogen peroxide (H2O2) to products that exhibit antimicrobial activity. LPO derived from bovine milk has found an application in food, cosmetics, and medical industries due to its structural and functional similarity to the human enzyme. Oral hygiene products enriched with the LPO system constitute an alternative to the classic fluoride caries prophylaxis. This review describes the physiological role of human salivary lactoperoxidase and compares the results of clinical trials and in vitro studies of LPO alone and complex dentifrices enriched with bovine LPO. The role of reactivators and inhibitors of LPO is discussed together with the possibility of using nanoparticles to increase the stabilization and activity of this enzyme.

Interference of carbidopa and other catechols with reactions catalyzed by peroxidases.

BACKGROUND: A number of compounds, including ascorbic acid, catecholamines, flavonoids, p-diphenols and hydrazine derivatives have been reported to interfere with peroxidase-based medical diagnostic tests (Trinder reaction) but the mechanisms of these effects have not been fully elucidated. METHODS: Reactions of bovine myeloperoxidase with o-dianisidine, bovine lactoperoxidase with ABTS and horseradish peroxidase with 4-aminoantipyrine/phenol in the presence of carbidopa, an anti-Parkinsonian drug, and other catechols, including l-dopa, were monitored spectrophotometrically and by measuring hydrogen peroxide consumption. RESULTS: Chromophore formation in all three enzyme/substrate systems was blocked in the presence of carbidopa and other catechols. However, the rates of hydrogen peroxide consumption were not much affected. Irreversible enzyme inhibition was also insignificant. CONCLUSIONS: Tested compounds reduced the oxidation products or intermediates of model substrates thus preventing chromophore formation. This interference may affect interpretation of results of diagnostic tests in samples from patients with Parkinson's disease treated with carbidopa and l-dopa. GENERAL SIGNIFICANCE: This mechanism allows prediction of interference in peroxidase-based diagnostic tests for other compounds, including drugs and natural products.

Expression profile of the CSF3 and LPO genes in milk from buffalo (Bubalus bubalis) with and without mastitis.

This study compared the expression profile of the candidate genes, CSF3 and LPO, by investigating the immune response mechanisms involved in the phenotype of resistance and susceptibility to mastitis of healthy and infected buffaloes. The Granulocyte Colony Stimulating Factor 3 (CSF3) and Lactoperoxidase (LPO) genes expression profiles were determined in 24 milk samples from buffaloes with (N = 12) and without (N = 12) mastitis, using the quantitative real-time PCR (qRT-PCR) technique. CSF3 and LPO expressions were 5.14 (P = 0.001) and 2.41 (P = 0.097) times higher in animals with mastitis compared to healthy animals, respectively, evidencing a trend toward different expressions of this gene in the studied groups. Our finding suggests that LPO and CSF3 genes are an important defense mechanism against mastitis in dairy buffaloes, and may be putative genes for selecting healthier animals in buffalo breeding programs.

Clinical concentrations of peroxidases cause dysbiosis in in vitro oral biofilms.

BACKGROUND AND OBJECTIVE: Little is known about the initiation of dysbiosis in oral biofilms, a topic of prime importance for understanding the etiology of, and preventing, periodontitis. The aim of this study was to evaluate the effect of different concentrations of crevicular and salivary peroxidase and catalase on dysbiosis in multispecies biofilms in vitro. MATERIAL AND METHODS: The spotting technique was used to identify the effect of different concentrations of myeloperoxidase, lactoperoxidase, erythrocyte catalase, and horseradish peroxidase in salivary and crevicular fluid on the inhibitory effect of commensals on pathobiont growth. Vitality-quantitative real-time PCR was performed to quantify the dysbiotic effect of the peroxidases (adjusted to concentrations found in periodontal health, gingivitis, and periodontitis) on multispecies microbial communities. RESULTS: Agar plate and multispecies ecology experiments showed that production of hydrogen peroxide (H2 O2 ) by commensal bacteria decreases pathobiont growth and colonization. Peroxidases at concentrations found in crevicular fluid and saliva neutralized this inhibitory effect. In multispecies communities, myeloperoxidase, at the crevicular fluid concentrations found in periodontitis, resulted in a 1-3 Log increase in pathobionts when compared with the crevicular fluid concentrations found in periodontal health. The effect of salivary lactoperoxidase and salivary myeloperoxidase concentrations was, in general, similar to the effect of crevicular myeloperoxidase concentrations. CONCLUSIONS: Commensal species suppress pathobionts by producing H2 O2 . Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to dysbiosis by allowing the outgrowth of pathobionts.

Immobilization of lactoperoxidase on graphene oxide nanosheets with improved activity and stability.

OBJECTIVES: The purpose of this study was to develop a facile and efficient method to enhance the stability and activity of lactoperoxidase (LPO) by using its immobilization on graphene oxide nanosheets (GO-NS). METHODS: Following the LPO purification from bovine whey, it was immobilized onto functionalized GO-NS using glutaraldehyde as cross-linker. Kinetic properties and stability of free and immobilized LPO were investigated. RESULTS: LPO was purified 59.13 fold with a specific activity of 5.78 U/mg protein. The successful immobilization of LPO on functionalized GO-NS was confirmed by using dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The overall results showed that the stability of the immobilized LPO was considerably improved compared to free LPO. Apparent Km and Vmax of LPO also indicated that the immobilized enzyme had greater affinity to the substrate than the native enzyme. CONCLUSIONS: Graphene oxide nanosheets are effective means for immobilization of LPO.

Reaction Product Variability and Biological Activity of the Lactoperoxidase System Depending on Medium Ionic Strength and pH, and on Substrate Relative Concentration.

The potential of ions produced in water by the lactoperoxidase system against plant pests has shown promising results. We tested the bioactivity of ions produced by the lactoperoxidase oxidation of I- and SCN- in several buffers or in tap water and characterized the ions produced. In vitro biological activity was tested against Penicillium expansum, the causal agent of mold in fruits, and the major cause of patulin contamination of fruit juices and compotes. In buffers, the ionic concentration was increased 3-fold, and pathogen inhibition was obtained down to the 1:15 dilution. In tap water, the ionic concentration was weaker, and pathogen inhibition was obtained only down to the 1:3 dilution. Acidic buffer increased ion concentrations as compared to less acidic (pH 5.6 or 6.2) or neutral buffers, as do increased ionic strength. 13 C-labelled SCN- and MS showed that different ions were produced in water and in buffers. In specific conditions the ion solution turned yellow and a product was formed, probably diiodothiocyanate (I2 SCN- ), giving an intense signal at 49.7 ppm in 13 C-NMR. The formation of the signal was unambiguously favored in acidic media and disadvantaged or inhibited in neutral or basic conditions. It was enhanced at a specific SCN- : I- ratio of 1:4.5, but decreased when the ratio was 1:2, and was inhibited at ratio SCN- >I- . We demonstrated that the formation of the signal required the interaction between I2 and SCN- , and MS showed the presence of I2 SCN- .

Lactoperoxidase immobilization on silver nanoparticles enhances its antimicrobial activity.

Lactoperoxidase (LPO) is an antimicrobial protein present in milk that plays an important role in natural defence mechanisms during neonatal and adult life. The antimicrobial activity of LPO has been commercially adapted for increasing the shelf life of dairy products. Immobilization of LPO on silver nanoparticles (AgNPs) is a promising way to enhance the antimicrobial activity of LPO. In the current study, LPO was immobilized on AgNPs to form LPO/AgNP conjugate. The immobilized LPO/AgNP conjugate was characterized by various biophysical techniques. The enhanced antibacterial activity of the conjugate was tested against E. coli in culture at 2 h intervals for 10 h. The results showed successful synthesis of spherical AgNPs. LPO was immobilized on AgNPs with agglomerate sizes averaging approximately 50 nm. The immobilized conjugate exhibited stronger antibacterial activity against E. coli in comparison to free LPO. This study may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO system in dairy and other industries.

Antiviral activity of hypothiocyanite produced by lactoperoxidase against influenza A and B viruses and mode of its antiviral action.

Hypothiocyanite (OSCN-) is a natural component of human saliva and is produced by the lactoperoxidase (LPO)/thiocyanate/hydrogen peroxide (H2O2) system. OSCN- has been previously shown to exhibit antiviral activity against influenza viruses (IFV) A/H1N1/2009 and A/H1N2/2009 in vitro as well as antimicrobial and antifungal activities. We elucidated the antiviral activity of OSCN- against both IFV types A and B and the mode of its antiviral action. OSCN- was produced constantly at 900 ± 200 µmol/l in Na3PO4 buffer solution containing NaSCN and LPO in the presence of H2O2 as an original OSCN- solution. In a plaque reduction assay, IFV A/PR/8/34 (H1N1), A/Fukushima/13/43 (H3N2), B/Singapore/222/97, and B/Fukushima/15/93 were exposed to various concentrations of OSCN- for 0 to 30 min before adsorption to MDCK cells, and plaque formation was examined. OSCN- exhibited significant similar antiviral activities against all four viruses without cytotoxicity, and the EC50 values for them were from 57 ± 16 to 148 ± 27 µmol/l regardless of the exposure times. The exposure of MDCK cells to OSCN- before viral adsorption did not affect its anti-IFV activity (EC50: more than 450 µmol/l), but the exposure after viral adsorption affected it moderately (EC50: 380 ± 40 µmol/l). Moreover, the exposure of virus particles to OSCN- at 450 µmol/l did not affect the hemagglutinin activity of IFV in hemagglutination inhibition assay. These results suggest that the attachment of OSCN- to the viral envelope critically contributes to the mode of antiviral action of OSCN- without interfering with viral adsorption. Keywords: hypothiocyanite; influenza virus type A; influenza virus type B; lactoperoxidase; antiviral activity.