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L-Acetylcarnitine (ALC), a versatile compound, has demonstrated beneficial effects in depression, Alzheimer's disease, cognitive impairment, and other conditions. This study focuses on its antithyroid activity. The precursor molecule, L-carnitine, inhibited the uptake of triiodothyronine (T3) and thyroxine (T4), and it is possible that ALC may reduce the iodination process of T3 and T4. Currently, antithyroid drugs are used to control the excessive production of thyroid hormones (TH) through various mechanisms: (i) forming electron donor-acceptor complexes with molecular iodine, (ii) eliminating hydrogen peroxide, and (iii) inhibiting the enzyme thyroid peroxidase. To understand the pharmacological properties of ALC, we investigated its plausible mechanisms of action. ALC demonstrated the ability to capture iodine (K c  = 8.07 ± 0.32 x 10 5 M -1 ), inhibit the enzyme lactoperoxidase (LPO) (IC 50  = 17.60 ± 0.76 µM), and scavenge H 2 O 2 (39.82 ± 0.67 mM). A comprehensive physicochemical characterization of ALC was performed using FTIR, Raman, and UV-Vis spectroscopy, along with theoretical DFT calculations. The inhibition process was assessed through fluorescence spectroscopy and vibrational analysis. Docking and molecular dynamics simulations were carried out to predict the binding mode of ALC to LPO and to gain a better understanding into the inhibition process. Furthermore, albumin binding experiments were also conducted. These findings highlight the potential of ALC as a therapeutic agent, providing valuable insights for further investigating its role in the treatment of thyroid disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

There is an urgent need in the medicinal fields to discover biocompatible nanoformulations with low cytotoxicity, which provide new strategies for promising therapies for several types of tumors. Bovine lactoperoxidase (LP) and lactoferrin (LF) have recently attracted attention in medicine for their antitumor activities with recognized safety pattern. Both LP and LF are suitable proteins to be coated or adsorbed to Cu and Fe nanometals for developing stable nanoformulations that boost immunity and strong anticancer effects. New nanometals of Cu and Fe NPs embedded in LP and LF forming novel nanocombinations of LP-CNPs and LF-FNPs had a spherical shape with an average nanosize of about 21 nm. The combination of LP-CNPs and LF-FNPs significantly exhibited the highest growth inhibitory efficacy, in terms of effectively lowering the half-maximal inhibitory concentration (IC 50 ) values, against Caco-2, HepG2 and MCF7 cells comparing to nanometals, LP, LF and individual nanoproteins (LP-CNPs or LF-FNPs). The highest apoptotic effect of this nanocombination (LP-CNPs and LF-FNPs) was confirmed by the highest percentages of annexin-stained apoptotic cells and G0 population with the strongest alteration in the expression of two well-characterized apoptosis guards (p53 and Bcl-2) and the maximum suppression in the proliferation marker (Ki-67). Also, the in silico analysis predicted that LP-CNPs and LF-FNPs enhanced AMP-activated protein kinase (AMPK, p53 activator) activity and inhibited cancer migration-related proteases (cathepsin B and matrix metalloproteinase (MMP)-9). Our results offer for the first time that these novel nanocombinations of LP and LF were superior in their selectivity and apoptosis-mediating anticancer activity to Cu and Fe nanometals as well as the free form of these proteins or their individual nanoforms., (© 2022. The Author(s).)

Mouthwash is a commonly used product and has been proposed as an alternative intervention to prevent gonorrhea transmission. However, the long-term effects of mouthwash on the oral microbiota are largely unknown. We investigated the impact of 12 weeks of daily mouthwash use on the oropharyngeal microbiota in a subset of men who have sex with men who participated in a randomized trial comparing the efficacy of two alcohol-free mouthwashes for the prevention of gonorrhea. We characterized the oropharyngeal microbiota using 16S rRNA gene sequencing of tonsillar fossae samples collected before and after 12 weeks of daily use of Listerine mouthwash or Biotène dry mouth oral rinse. Permutational multivariate analysis of variance (PERMANOVA) was used to assess differences in oropharyngeal microbiota composition following mouthwash use. Differential abundance testing was performed using ALDEx2, with false-discovery rate correction. A total of 306 samples from 153 men were analyzed (Listerine, n  = 78 and Biotène, n  = 75). There was no difference in the overall structure of the oropharyngeal microbiota following Listerine or Biotène use (PERMANOVA P  = 0.413 and P  = 0.331, respectively). Although no bacterial taxa were significantly differentially abundant following Listerine use, we observed a small but significant decrease in the abundance of both Streptococcus and Leptotrichia following Biotène use. Overall, our findings suggest that daily use of antiseptic mouthwash has minimal long-term effects on the composition of the oropharyngeal microbiota. IMPORTANCE Given the role of the oral microbiota in human health, it is important to understand if and how external factors influence its composition. Mouthwash use is common in some populations, and the use of antiseptic mouthwash has been proposed as an alternative intervention to prevent gonorrhea transmission. However, the long-term effect of mouthwash use on the oral microbiota composition is largely unknown. We found that daily use of two different commercially available mouthwashes had limited long-term effects on the composition of the oropharyngeal microbiota over a 12-week period. The results from our study and prior studies highlight that different mouthwashes may differentially affect the oral microbiome composition and that further studies are needed to determine if mouthwash use induces short-term changes to the oral microbiota that may have detrimental effects.

Milk consumption may modify the risk of squamous cell carcinoma. The role of milk to modulate the gene expression in oral squamous cell carcinoma cells has not been investigated so far. Here, HSC2 oral squamous carcinoma cells were exposed to an aqueous fraction of human milk and a whole-genome array was performed. Among the genes that were significantly reduced by human and cow milk were the DNA-binding protein inhibitor 1 (ID1), ID3 and Distal-Less Homeobox 2 (DLX2) in HSC2 cells. Also, in TR146 oral squamous carcinoma cells, there was a tendency towards a decreased gene expression. Upon size fractionation, lactoperoxidase but not lactoferrin and osteopontin was identified to reduce ID1 and ID3 in HSC2 cells. Dairy products and hypoallergenic infant formula failed to decrease the respective genes. These data suggest that milk can reduce the expression of transcription factors in oral squamous carcinoma cells.

Interaction between nanoparticles (NPs) and protein is particularly important due to the formation of dynamic nanoparticle-protein complex. The current study indicated that silica NPs were able to induce conformational modification in the adsorbed lactoperoxidase (LPO) which in turns degrades the synthetic dyes. The maximum degradation efficiency was recorded for the LPO modified silica NPs in the presence of H 2 O 2 comparing with either free LPO or silica NPs. Degradation efficiency of crystal violet and commassie blue R250 after 6 h was assessed to be 100(%). Also, degradation efficiency of Congo red reached 90.6% and 79.3% in the presence and absence of H 2 O 2 , respectively, however methyl red degradation efficiency recorded 85%. The viability assay experiment indicated that the IC 50 value of the LPO modified silica NPs on human fibroblast cells reached 2.8 mg/ml after 48 h incubation. In addition to dye removal, the LPO modified silica NPs were able to inhibit the antibiotic resistant bacterial strains (Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp.) at concentrations up to 2.5 mg/ml with inhibition activity about 95%. These findings emphasized that the ability of LPO for degradation of the synthetic dyes after adsorption on silica NPs besides it could be a promising agent with potent inhibitory effect targeting a wide range of multidrug resistant bacteria.

Although partial thickness burns are the most frequently reported burn injuries, there is no consensus on the optimal treatment. The objective of this study was to compare the clinical effectiveness and scar quality of Flaminal® Forte to silver sulfadiazine (Flamazine®) in the treatment of partial thickness burns. In this two-arm open label multicenter randomized controlled trial, adult patients with acute partial thickness burns and an affected total body surface area of less than 30% were randomized between Flaminal® Forte and Flamazine® and followed for 12 months. Dressing changes in the Flamazine® group were performed daily, and in the Flaminal® group during the first 3 days post burn and thereafter every other day until complete wound healing or surgery. Forty-one patients were randomly allocated to Flaminal® Forte and 48 patients to Flamazine®. The primary outcome was time to wound healing, which did not differ between the groups: median 18 days with Flaminal® Forte (range 8-49 days) versus 16 days with Flamazine® (range 7-48 days; p = 0.24). Regarding the secondary outcomes during hospital admission, there were no statistically significant differences between the groups concerning need for surgery, pain scores, pruritus, or pain-related and anticipatory anxiety. More patients in the Flaminal® group developed wound colonization (78% versus 32%, p < 0.001), but the treatment groups did not differ regarding the incidence of local infections and use of systemic antibiotics. In terms of scar quality, no statistically significant differences between both treatment groups were found regarding subjective scar assessment (Patient and Observer Scar Assessment Scale (POSAS)), scar melanin and pigmentation (DermaSpectrometer®), and scar elasticity and maximal extension (Cutometer®) during 12 month postburn. In conclusion, time to wound healing did not differ, but the use of Flaminal® Forte seemed favorable because less dressing changes are needed which lowers the burden of wound care., (© 2019 The Authors. Wound Repair and Regeneration published by Wiley Periodicals, Inc. on behalf of by the Wound Healing Society.)

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.

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.

The NOX/DUOX family of NADPH oxidases are transmembrane proteins generating reactive oxygen species as their primary enzymatic products. NADPH oxidase (NOX) 1-5 and Dual oxidase (DUOX) 1 and 2 are members of this family. These enzymes have several biological functions including immune defense, hormone biosynthesis, fertilization, cell proliferation and differentiation, extracellular matrix formation and vascular regulation. They are found in a variety of tissues such as the airways, salivary glands, colon, thyroid gland and lymphoid organs. The discovery of NADPH oxidases has drastically transformed our view of the biology of reactive oxygen species and oxidative stress. Roles of several isoforms including DUOX1 and DUOX2 in host innate immune defense have been implicated and are still being uncovered. DUOX enzymes highly expressed in the respiratory and salivary gland epithelium have been proposed as the major sources of hydrogen peroxide supporting mucosal oxidative antimicrobial defenses. In this review, we shortly present data on DUOX discovery, structure and function, and provide a detailed, up-to-date summary of discoveries regarding antibacterial, antiviral, antifungal, and antiparasitic functions of DUOX enzymes. We also present all the literature describing the immune functions of lactoperoxidase, an enzyme working in partnership with DUOX to produce antimicrobial substances.

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 (H 2 O 2 ) 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 H 2 O 2 . Catalase and peroxidases, at clinically relevant concentrations, can neutralize this effect and thereby can contribute to dysbiosis by allowing the outgrowth of pathobionts., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Bovine lactoperoxidase (LPO) and lactoferrin (LF) are suitable proteins to be loaded or adsorbed to chitosan nanoparticles (NPs) for preparing stable nanoformulations with potent anticancer activity. In the present study, nanocombinations of LPO and LF revealed improvement in their stability and activity compared to single (free or nanoformulated) bovine proteins. The coating or loading of LPO-loaded NPs with LF resulted in the highest synergistic cytotoxicity effect against Caco-2, HepG-2, MCF-7 and PC-3 cells in comparison with other NPs and free proteins without causing toxicity toward normal cells. This synergistic improvement in the anticancer activity was apoptosis-dependent that was confirmed by severe alterations in cellular morphology, high percentage of annexin-stained cells and sub-G1 populations as well as nuclear staining with orange fluorescence of treated cancer cells. Additionally, significant alterations in the expression of well characterized cellular proliferation and apoptosis guards (NF-κB, Bcl-2 and p53) in these NPs-treated cancer cells compared to 5-fluorouracil (5-FU) treated cells. Our findings provide for the first time that these new synergistic nanoformulated forms of LPO and LF were superior in their selective apoptosis-mediating anticancer effect than free form of these proteins and 5-FU. LF coating or loading of LPO-loaded NPs present as promising therapy for cancer.

The oral microbiota influences health and disease states. Some gram-negative anaerobic bacteria play important roles in tissue destruction associated with periodontal disease. Lactoferrin (LF) and lactoperoxidase (LPO) are antimicrobial proteins found in saliva; however, their influence on the whole oral microbiota currently remains unknown. In this randomized, double-blinded, placebo-controlled study, the effects of long-term ingestion of LF and LPO-containing tablets on the microbiota of supragingival plaque and tongue coating were assessed. Forty-six older individuals ingested placebo or test tablets after every meal for 8 weeks. The relative abundance of bacterial species was assessed by 16S rRNA gene high-throughput sequencing. Most of the bacterial species in supragingival plaque and tongue coating that exhibited significant decreases in the test group were gram-negative bacteria, including periodontal pathogens. Decreases in the total relative abundance of gram-negative organisms in supragingival plaque and tongue coating correlated with improvements in assessed variables related to oral health, such as oral malodor and plaque accumulation. Furthermore, there was significantly less microbiota diversity in supragingival plaque at 8 weeks in the test group than in the placebo group and low microbiota diversity correlated with improvements in assessed variables related to oral health. These results suggest that LF and LPO-containing tablets promote a shift from a highly diverse and gram-negative-dominated to a gram-positive-dominated community in the microbiota of supragingival plaque and tongue coating. This microbial shift may contribute to improvements in oral health, including oral malodor and state of the gingiva., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

The increasing occurrence of multidrug resistant bacteria causing bacteremia infection, constitutes a major health problem, difficult-to-treat bacteremia due to its ability to form biofilm. Buffalo milk lactoperoxidase (BMLpo) is effective and safe to use as bacteriostatic agent. The MIC of BMLpo and amikacin were used to evaluate the antibiofilm activity against resistant L. monocytogenes and S. typhi. Prophylactic effects of BMLpo against L. monocytogenes and S. typhi bacteremia in vivo have been tested and ELISA test used to evaluate serum cytokines. Significant antibiofilm activity of BMLpo observed against the highest biofilm producer isolates. Our results showed that the prophylactic effect of BMLpo in BALB/c mice bacteremic model. A significant clearance of L. monocytogenes and S. typhi, investigated in blood and different organs tissues in BMLpo-treated infected groups when compared to the non-treated groups. Further, analysis of serum cytokines levels revealed that BMLpo prophylaxis modulates their release in different way when it compared to the control. This study showed, BMLpo effects as an alternative antibiofilm agent to compact gram negative pathogens, and protects the host against bacteremia infection. Moreover, the BMLpo role as an immunomodulatory. These investigations indicated the BMLpo crucial role in the practical clinical applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 H 2 O 2 , 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%.

Aim: Lactoferrin and lactoperoxidase have antimicrobial effects against oral pathogens. This randomized, double-blinded, placebo-controlled parallel group study tested the efficacy of a lactoferrin and lactoperoxidase-containing tablet (LF + LPO tablet) in improving the oral hygiene status of older individuals., Methods: A total of 46 participants (31 nursing home residents and 15 healthy older individuals) were randomly assigned to receive either lactoferrin and lactoperoxidase-containing tablets or placebo tablets, and were asked to suck on a tablet after every meal for 8 weeks. Oral and bacteriological assessments were carried out at baseline, 4 weeks and 8 weeks., Results: A total of 47 participants (test group n = 20; mean age 80.4 ± 6.4 years; placebo group n = 17; mean age 85.9 ± 6.7 years) were included in the efficacy analysis. In the test group, the total number of bacteria in the tongue coating was significantly reduced at 4 and 8 weeks compared with that at baseline, and the number of Porphyromonas gingivalis and Fusobacterium nucleatum was significantly reduced at 8 weeks. The total number of bacteria and the number of P. gingivalis in the supragingival plaque were significantly reduced at 8 weeks. Furthermore, there was a significant difference in the change in the number of P. gingivalis in supragingival plaque at 8 weeks between the two groups., Conclusions: Lactoferrin and lactoperoxidase-containing tablet ingestion showed antibacterial effects on periodontal bacteria present in the tongue coating and supragingival plaque, indicating that long-term ingestion could improve the oral hygiene of older individuals. Geriatr Gerontol Int 2017; 17: 714-721., (© 2016 Japan Geriatrics Society.)

This study compared the capacity of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) to that of a combination of lysozyme, lactoferrin, and lactoperoxidase (LLL) in root canal disinfectant for reducing the Streptococcus mutans counts from dentinal caries. Forty human permanent third molars were selected, and flat dentin surfaces were created. Carious lesions were induced using a microbiological model. The specimens were randomly divided into 2 groups (n = 20) according to the type of agent used: group 1, CPP-ACP; group 2, LLL. The S mutans counts were performed before application and after the first, second, and third applications of the agents. The duration of each application was 3 minutes. Carious dentin specimens were homogenized, diluted, and seeded onto mitis salivarius-bacitracin plates for viable counts of S mutans. Results showed that there was no significant reduction in the number of S mutans in group 1 after the applications of CPP-ACP (P > 0.05). In group 2, a significant reduction of S mutans was observed after the third application of LLL (P < 0.01). These results indicate that 3 applications of LLL enzymes can be used to reduce the number of S mutans in dentinal caries lesions.

Recent studies have demonstrated the effect of bleaching conditions and bleaching agent on flavor and functional properties of whey protein ingredients. Solids concentration at bleaching significantly affected bleaching efficacy and flavor effects of different bleaching agents. It is not known if these parameters influence quality of sweet whey powder (SWP). The purpose of this study was to determine the effects of solids concentration and bleaching agent on the flavor and bleaching efficacy of SWP. Colored cheddar whey was manufactured, fat separated, and pasteurized. Subsequently, the whey (6.7% solids) was bleached, concentrated using reverse osmosis (RO) to 14% solids, and then spray dried, or whey was concentrated before bleaching and then spray dried. Bleaching treatments included a control (no bleaching, 50 °C, 60 min), hydrogen peroxide (HP; 250 mg/kg, 50 °C, 60 min), benzoyl peroxide (50 mg/kg, 50 °C, 60 min), lactoperoxidase (20 mg/kg of HP, 50 °C, 30 min), and external peroxidase (MaxiBright, DSM Food Specialties, Delft, the Netherlands; 2 dairy bleaching units/mL, 50 °C, 30 min). The experiment was repeated in triplicate. Sensory properties and volatile compounds of SWP were evaluated by a trained panel and gas chromatography-mass spectrometry, respectively. Bleaching efficacy (norbixin destruction) and benzoic acid were measured by HPLC. Differences in bleaching efficacy, sensory and volatile compound profiles, and benzoic acid were observed with different bleaching agents, consistent with previous studies. Solids concentration affected bleaching efficacy of HP, but not other bleaching agents. The SWP from whey bleached with HP or lactoperoxidase following RO had increased cardboard and fatty flavors and higher concentrations of lipid oxidation compounds compared with SWP from whey bleached before RO. The SWP bleached with benzoyl peroxide after RO contained less benzoic acid than SWP from whey bleached before RO. These results indicate that solids concentration at bleaching and bleaching agent affect quality of SWP., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: As a result of consumers' concerns about chemicals there is a particular interest in the food industry to use natural bio-preservatives such as antimicrobial enzymes for antimicrobial packaging. Based on the antimicrobial activity of the lactoperoxidase system (LPOS), the present study evaluated the coating effect of LPOS incorporated into chitosan solution (CH) on the quality and shelf life extension of rainbow trout during refrigerated storage (4 ± 1 °C), for a period of 16 days., Results: The results indicated that samples of the CH+LPOS group had significantly lower numbers of Shewanella putrefaciens, Pseudomonas fluorescens, and psychrotrophic and mesophilic bacteria than did the CH and control group during the entire storage period. Total volatile basic nitrogen (TVB-N) levels for the CH+LPOS samples (22.07 mg 100 g(-1)) did not exceed the limit of consumption (30-35 mg N 100 g(-1)), while the CH (31.03 mg 100 g(-1) ) and control groups (37.78 mg 100 g(-1) ) reached this level at days 12 and 16, respectively. Thiobarbituric acid values of the CH and CH+LPOS samples, ranged between 0.49 and 0.51 on day 0 and 4.59-4.66 mg kg(-1) on day 16, were significantly lower (P < 0.05) than the corresponding values of the control samples (0.47 on day 0 to 4.78 mg kg(-1) on day 16 of storage) during the chilled storage period., Conclusion: The coating treatments (CH and CH+LPOS) extended the shelf life of trout fillets by at least 4 days as compared to the control samples, so that they showed moderate to high acceptability in all investigated sensory attributes even on the 16th day of storage., (© 2014 Society of Chemical Industry.)

This article continues a series of reviews on the abundance and roles of intrinsic disorder in milk proteins. Besides caseins, which are the major proteinaceous constituents of any milk that can be isolated by isoelectric precipitation, milk contains a set of soluble whey proteins, such as β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin, lactoperoxidase, glycomacropeptide, and proteose peptone (the last two are soluble casein derivatives). Lactoferrin and lactoperoxidase (LPO) are known to possess prominent biocidal activity, serving as efficient antibiotics and antiviral agents against a wide spectrum of bacteria, fungi, and viruses. LPO is a heme-containing peroxidase expressed as preproprotein. The mature protein has a single catalytic domain, structure of which is known for a protein isolated from several species. Functionally, LPO is a crucial component of the LPO system that includes LPO, hydrogen peroxide (H2O2), and thiocyanate (SCN(-)), being a well-studied, naturally occurring antimicrobial system in milk that is effective against many microorganisms and some viruses. Although various aspects of LPO structure and function are rather well studied and were subjects of several recent reviews, the abundance and potential functional roles of intrinsically disordered regions in this protein have never being addressed as of yet. The major goal of this article is to fill this gap and to show how intrinsic disorder is encoded in the amino acid sequence of LPO, and how intrinsic disorder is related to functions of this important milk protein.

Objectives: To investigate the effect of saliva substitutes on enamel erosion in vitro., Methods: A total of 204 bovine enamel samples were embedded in acrylic resin and allocated to 17 groups (n=12). The specimens were eroded in an artificial mouth (3 days; 6×30 s/days, flow rate: 2 ml/min) using citric acid (pH: 2.5). Immediately after the erosive attacks, saliva substitutes (12 sprays, 3 gels) were applied. Between the erosive cycles the specimens were rinsed with artificial saliva (flowrate: 0.5 ml/min). A SnCl2/AmF/NaF-containing mouthrinse was used as positive control, water spray served as negative control. Enamel loss was measured profilometrically and the data were analyzed using one-way ANOVA followed by Scheffé's post hoc tests (p<0.05)., Results: Four saliva substitutes increased enamel erosion, probably due to the low pH or the content of citric acid. Several saliva substitutes were able to reduce enamel erosion significantly by 60-90% (in the range of the positive control). The protective potential of these products was in the range of the positive control (reduction of enamel loss to 30% of negative control). The erosion-protective potential of these high-viscous products is probably related to their film-forming properties, leading to a mechanical protection of the surface., Conclusion: Saliva substitutes containing a very low pH exhibit a distinct erosive potential, while most high-viscous products present an erosion-protective effect. It can be recommended that patients suffering from xerostomia and at high risk for dental erosion should use high-viscous saliva substitutes, but should avoid saliva substitutes with low pH or containing citric acid., Clinical Significance: It can be recommended that patients suffering from xerostomia and at high risk for dental erosion should use high-viscous saliva substitutes, but should avoid saliva substitutes with low pH or containing citric acid., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The association of biofilms with wound chronicity has prompted a search for antimicrobial interventions that are effective against biofilms. A patented preparation of glucose oxidase, lactoperoxidase and guaiacol (GLG), which is the antibacterial component of Flaminal, has been shown to inhibit a wide range of bacteria, but it has not yet been tested on biofilms. This study aims to determine the effect of GLG on biofilms of Staphylococcus aureus, methicillin-resistant S. aureus and Pseudomonas aeruginosa. Static biofilms were grown in microtitre plates and on coverslips and treated with a range of concentrations of GLG. Effects were monitored by estimating biofilm biomass by staining with crystal violet, biofilm activity by staining with either resazurin or fluorescein diacetate and biofilm viability by staining with LIVE/DEAD BacLight Bacterial Viability Kit. GLG was able to prevent the formation of biofilms at concentration ≤0.5% (w/v) and higher concentrations were required to inhibit established biofilms. GLG did not disrupt biofilm biomass. Staphylococci were more susceptible to GLG than P. aeruginosa. These in vitro findings must be verified by in vivo studies., (© 2013 The Author. International Wound Journal © 2013 John Wiley & Sons Ltd and Medicalhelplines.com Inc.)

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.)

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-β2 (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.

Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically bleached WPC80, lactoperoxidase-bleached WPC80 contained higher relative abundance of 2,3-octadienone, 2-pentyl furan, and hexanal than those bleached with added EP. Bleach times, bleaching efficacy, and flavor results suggest that enzymatic bleaching may be a viable and desirable alternative to HP bleaching of fluid whey or retentate., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. 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.)

Lactoperoxidase (LP) is the second most abundant enzyme in bovine milk and has been used in conjunction with hydrogen peroxide (H₂O₂) and thiocyanate (SCN⁻) to work as an antimicrobial in raw milk where pasteurization is not feasible. Thiocyanate is naturally present and the lactoperoxidase system purportedly can be used to bleach dairy products, such as whey, with the addition of very little H₂O₂ to the system. This study had 3 objectives: 1) to quantify the amount of H₂O₂ necessary for bleaching of fluid whey using the LP system, 2) to monitor LP activity from raw milk through manufacture of liquid whey, and 3) to compare the flavor of whey protein concentrate 80% (WPC80) bleached by the LP system to that bleached by traditional H₂O₂ bleaching. Cheddar cheese whey with annatto (15 mL of annatto/454 kg of milk, annatto with 3% wt/vol norbixin content) was manufactured using a standard Cheddar cheesemaking procedure. Various levels of H₂O₂ (5-100 mg/kg) were added to fluid whey to determine the optimum concentration of H₂O₂ for LP activity, which was measured using an established colorimetric method. In subsequent experiments, fat-separated whey was bleached for 1h with 250 mg of H₂O₂/kg (traditional) or 20 mg of H₂O₂/kg (LP system). The WPC80 was manufactured from whey bleached with 250 mg of H₂O₂/kg or 20mg of H₂O₂/kg. All samples were subjected to color analysis (Hunter color values and norbixin extraction) and proximate analysis (fat, protein, and moisture). Sensory and instrumental volatile analyses were conducted on WPC80. Optimal LP bleaching in fluid whey occurred with the addition of 20mg of H₂O₂/kg. Bleaching of fluid whey at either 35 or 50°C for 1 h with LP resulted in > 99% norbixin destruction compared with 32 or 47% destruction from bleaching with 250 mg of H₂O₂/kg, at 35 or 50°C for 1 h, respectively. Higher aroma intensity and increased lipid oxidation compounds were documented in WPC80 from bleached whey compared with WPC80 from unbleached whey. Monitoring of LP activity throughout cheese and whey manufacture showed that LP activity sharply decreased after 30 min of bleaching (17.01 ± 1.4 to < 1 U/mL), suggesting that sufficient bleaching takes place in a very short amount of time. Lactoperoxidase averaged 13.01 ± 0.7 U/mL in unpasteurized, fat-separated liquid whey and 138.6 ± 11.9 U/mL in concentrated retentate (11% solids). Lactoperoxidase may be a viable alternative for chemical whey bleaching., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

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 fungistatic and fungicidal activity of hyaluronic acid (HA) and the influences of HA on the anticandidal activities of lysozyme and the peroxidase system., Materials and Methods: HA, hen egg-white lysozyme, and the bovine lactoperoxidase system were used. Candida albicans ATCC 10231, 18804, and 11006 strains were used in the experiments. The fungistatic activity of HA was determined by measuring the optical densities of the cultures. The candidacidal activity of HA and the influences of HA on the candidacidal activities of lysozyme and the peroxidase system were determined by comparing the numbers of colony-forming units., Results: Hyaluronic acid displayed inhibitory effects on the growth of C. albicans, and the inhibitory effects were proportional to HA concentration. HA did not have any measurable candidacidal activity. HA showed inhibitory effects on the candidacidal activities of lysozyme, and the peroxidase system that was proportional to HA concentration. HA at 1.0-2.0 mg ml(-1) almost completely inhibited the candidacidal activities of lysozyme and the peroxidase system., Conclusions: Hyaluronic acid possesses fungistatic activity but no candidacidal activity. HA showed inhibitory effects on the candidacidal activities of lysozyme and the peroxidase system., (© 2011 John Wiley & Sons A/S.)

Products have been developed to provide palliation for persons with dry mouth. In addition to mouth-wetting agents, some products incorporate antimicrobial constituents with the goal of improving oral microbial defenses. The aim of this in vitro study was to investigate the potential antimicrobial and antifungal effects of two commercially available saliva substitutes on Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans by using the agar-well diffusion method. Antimicrobial activity as measured by the size of the inhibition zone growth for S. mutans and L. acidophilus was observed only with Biotene Dry Mouth Oral Rinse® and BioXtra® gel. The zone of inhibition of Biotene Dry Mouth Oral Rinse was larger than that of BioXtra gel (p= 0.00, p < 0.01). No anticandidal effect was seen with any of the test products. The pH of the preparations, the variations between the amount of active ingredients within the products, and the potential antimicrobial effects of inactive ingredients should be investigated to determine the factors that impacted microbial inhibition., (© 2011 Special Care Dentistry Association and Wiley Periodicals, Inc.)

A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cross-protection of acid-adapted Escherichia coli O157:H7 against inimical stresses is mediated by the glucose-repressed sigma factor RpoS. However, many food systems in which E. coli O157:H7 occurs are complex and contain glucose. This study was aimed at investigating the contribution of acid and lactoperoxidase (LP)-inducible genes to cross-protection of E. coli O157:H7 against LP system and lactic acid (LA) in Tryptone Soy Broth (TSB). Acid-adapted and non-adapted E. coli O157:H7 were challenged to activated LP and LA at pH 4.0 and 5.0 in TSB for 6h at 25°C followed by expression of acid and LP-inducible genes. Acid-adapted E. coli showed cross-protection against activated LP and LA. All the acid-inducible genes tested were repressed at pH 4.0 with or without activated LP system. At pH 7.4, gadA, ompC and ompF were induced in acid-adapted cells. Induction of corA occurred in non-adapted cells but was repressed in acid-adapted cells. Although acid-inducible genes were repressed at pH 4.0, high resistance of acid-adapted cells indicates that expression of acid-inducible genes occurred during acid adaptation and not the actual challenge. Repression of rpoS indicates that RpoS-independent systems contribute to cross-protection in acid-adapted E. coli O157:H7., (2009 Elsevier GmbH. 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.)

Bovine lactoperoxidase (LPO) was purified from skimmed milk using amberlite CG-50-H+ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography. Lactoperoxidase was purified 20.45-fold with a yield of 28.8%. Purity of enzyme checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and a single band was observed. Km was 0.25 mM at 20 degrees C, Vmax value was 7.95 micromol/ml min at 20 degrees C (pH 6.0). Antibacterial study was done by disk diffusion method of Kir-by-Bauer using Mueller-Hinton agar medium with slight modification. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate-100 mM H2O2 medium for some bacteria (Brevibacillus centrosaurus, B. choshinensis, B. lyticum, Cedecea davisae, Chryseobacterium indoltheticum, Clavibacter michiganense pv. insidiosum, Kocuria erythromyxa, K. kristinae, K. rosea, K. varians, Paenibacillus validus, Pseudomonas syringae pv. populans, Ralstonia pickettii, Rhodococcus wratislaviensis, Serratia fonticola, Streptomyces violaceusniger, Vibrio cholerae-nonO1) respectively, and compared with well known antibacterial substances (levofloxacin, netilmicin). LPO system has inhibition effects on all type bacteria and concentration is really important such as LPO-100 mM thiocyanate-100 mM H2O2 system was proposed as an effective agent against many factors causing several diseases.

Background: The positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H2O2 on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H2O2 system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H2O2 combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min., Results: The bactericidal and fungicidal effects of lactoperoxidase on Streptococcus mutans and sanguinis and Candida albicans were evaluated by using two test mixtures of a 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide solution, one without and one with lactoperoxidase. Following the quantitative suspension tests (EN 1040 and EN 1275), the growth of surviving bacteria and fungi in a nutrient broth was measured. The reduction factor in the suspension test without lactoperoxidase enzyme was < 1 for all three tested organisms. Thus, the mixtures of 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide had no in vitro antimicrobial effect on Streptococcus mutans and sanguinis or Candida albicans. However, the suspension test with lactoperoxidase showed a high bactericidal and fungicidal effectiveness in vitro., Conclusion: The tested thiocyanate and H2O2 mixtures showed no relevant antimicrobial effect. However, by adding lactoperoxidase enzyme, the mixtures became not only an effective bactericidal (Streptococcus mutans and sanguinis) but also a fungicidal (Candida albicans) agent.

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.

In this study, the antimicrobial effect of lactoperoxidase (LPS) incorporated alginate films was investigated on Escherichia coli (NRRL B-3008), Listeria innocua (NRRL B-33314), and Pseudomonas fluorescens (NRRL B-253) in presence of different concentrations of H(2)O(2) (0.2, 0.4, and 0.8 mM) and KSCN (1, 2, and 4 mM). The incorporation of 70 nmol ABTS/min/cm(2) LPS into alginate films gave 0.66 to 0.85 nmol ABTS/min/cm(2) enzyme activity at 0.2 to 0.8 mM H(2)O(2) concentration range. The antimicrobial activity of LPS system on target bacteria changed according to the concentrations of KSCN and H(2)O(2). The growth of all tested bacteria was prevented for a 6-h period by applying LPS system in presence of 0.4 or 0.8 mM H(2)O(2) and 4 mM KSCN. At 0.8 mM H(2)O(2) and 4 mM KSCN, the LPS system also inhibited growth of L. innocua and P. fluorescens for a 24-h incubation period, whereas E. coli growth could not be inhibited for 24 h under these conditions. At 0.2 mM H(2)O(2) and 1 to 4 mM KSCN, a considerable inhibitory effect was obtained only on P. fluorescens. The decreasing order of the resistance of studied bacteria to LPS system is as follows: E. coli, L. innocua, and P. fluorescens. The developed antimicrobial system has a good potential for use in meat, poultry, and seafood since alginate coatings are already used in these products. Further studies are needed to test the LPS incorporated edible films in real food systems.

The effect of lactoperoxidase (LPO) on dextran sulfate sodium-induced colitis was examined in mice. After 9 d of colitis induction, weight loss, colon shortening, and the histological score were significantly suppressed in mice orally administered LPO (62.5 mg/body/d) as compared to a group administered bovine serum albumin. These results suggest that LPO exhibits anti-inflammatory effects in the gastrointestinal tract.

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.

The inhibitory activity of nisin (N), reuterin (R), and the lactoperoxidase system (LPS), added individually or in combination, against Listeria monocytogenes and Staphylococcus aureus was investigated in "cuajada" (curdled milk), a semisolid dairy product manufactured in Spain. Cuajada was manufactured from UHT skim milk separately inoculated with L. monocytogenes and Staph. aureus, each at approximately 4 log cfu/mL, and held under conditions of temperature abuse (10 degrees C). On d 3, a synergistic bactericidal activity was observed for the combinations of biopreservatives assayed, with L. monocytogenes counts of only 0.30 log cfu/mL in cuajada made with N + R + LPS vs. 8.31 log cfu/mL in control cuajada. After 12 d, L. monocytogenes could not be detected in cuajada made with added N + LPS or N + R + LPS. Staphylococcus aureus was more resistant than L. monocytogenes to biopreservatives added individually. On d 3, the synergistic effect of the 3 biopreservatives against Staph. aureus resulted in counts of 3.03 log cfu/mL in cuajada made with N + R + LPS vs. 6.40 in control cuajada. After 12 d, Staph. aureus counts were 2.61 log cfu/mL in cuajada made with N + R + LPS, whereas they ranged from 6.11 to 7.70 log cfu/mL in control cuajada and in cuajada made with other combinations of biopreservatives. The most pronounced decrease in pathogen counts was achieved by the triple combination N + R + LPS, which acted synergistically on the inactivation of L. monocytogenes and Staph. aureus in cuajada over 12 d at 10 degrees C. The treatment combining these 3 natural biopreservatives at low concentrations, within the hurdle concept of food preservation, might be a useful tool to control the growth of pathogenic microorganisms in nonacidified dairy products.

Background: Oral and oropharyngeal decontamination is one of the main issues for preventing ventilator-associated pneumonia (VAP)., Objective: The objective of the study was to detect and compare in vitro antibacterial activities of 3 oral care products (OCP) against major VAP pathogens., Methods: Stabilized hydrogen peroxide (H(2)O(2)); 0.2% chlorhexidine gluconate (CHX); and a commercial product including glucose oxidase, lactoperoxidase, lysozyme, and lactoferrin (GLLL) were selected for this study. In total, 32 VAP isolates were studied by 2 different methods. Bacterial suspension was inoculated onto OCP-absorbed plates in the first method, and OCP was dropped onto bacteria inoculated plates in the second method. Two different bacterial suspensions were used as 10(-2) and 10(-4) dilutions of 0.5 McFarland turbidity., Results: In the first method, 6 (18%), 6 (18%), and 0 isolates in 10(-2) dilution and 13 (40%), 19 (59.3%), and 2 (6.2%) isolates in 10(-4) dilution of 0.5 McFarland bacterial turbidity were inhibited by CHX, H(2)O(2), and GLLL, respectively. In the second method, 31 (96.8%), 30 (93.7%), and 0 isolates in 10(-2) dilution and 32 (100%), 32 (100%), and 5 (15.6%) isolates in 10(-4) dilution were suppressed. In all dilutions and methods, antibacterial activity of CHX and H(2)O(2) were found more effective than GLLL against VAP pathogens (P < .05)., Conclusion: CHX and H(2)O(2) have good antibacterial effects against most isolated VAP pathogens in vitro. They could be suggested as oropharyngeal decontamination agents for reducing VAP incidence.

Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.

Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.

Milk and colostrum are rich in proteins and peptides which play a crucial role in development of the immune system in mammalian offspring. Immunotropic properties of these compounds prompted investigators to search for their utility in prevention and therapy of various disorders in humans. The following constituents of milk are of particular interest: 1) Lactoferrin (LF)--exhibits antibacterial, antifungal, antiviral, antiparasite and antitumor activities. It is protective with regard to intestinal epithelium, promotes bone growth and accelerates recovery of the immune system function in immunocompromised animal; 2) A Proline-Rich Polypeptide (PRP) shows a variety of immunotropic functions, including promotion of T-cell maturation and inhibition'of autoimmune disorders. PRP was recently found to improve or stabilize the Instrumental Activity of Daily Living status in Alzheimer's disease patients. 3) Casein--has been protective in experimental bacteremia by eliciting myelopoiesis. Casein hydrolyzates were also protective in diabetic animals, reduced the tumor growth and diminished colicky symptoms in infants. Casein-derived peptides have been found to have antihypertensive effects. Glycomacropeptide (GMP)--a peptide derived from kappa casein, exhibits antibacterial and antithrombotic activities. 4) Alpha lactalbumin (LA)--demonstrates antiviral, antitumor and anti-stress properties. LA-enriched diets were anxiolytic, lowered blood pressure in rats, prevented diarrhea and led to a better weight gain in malnourished children. 5) Lysozyme--is effective in treatment of periodentitis and prevention of tooth decay. Milk enriched in lysozyme was used in feeding premature infants suffering from concomitant diseases. 6) Lactoperoxidase--shows antibacterial properties. In conclusion, milk-derived proteins and peptides are bio-accessible and safe for the prevention and treatment of numerous disorders in humans.

Toxoplasma gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasma was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose-glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T. gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.

Wound bed preparation remains a very important issue in wound healing. To promote the production of granulation tissue, it is necessary to remove necrotic tissue and to control infection. Necrotic tissue may be removed using a hydrogel preparation. Flaminal and Flaminal Hydro (Flen Pharma, Belgium) are 2 new hydroactive colloid gel dressings with state antibacterial properties. These properties are attributed to an enzymatic complex in their formulation. In the study described in this report, the antibacterial effects of Flaminal and Flaminal Hydro were confirmed in an in vitro as well as an in vivo setting. It was also demonstrated that Flaminal and Flaminal Hydro are not toxic to keratinocytes in vitro using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test.

The effects of whey protein isolate (WPI) coatings incorporating a lactoperoxidase system (LPOS) on the inhibition of Salmonella enterica and Escherichia coli O157:H7 on roasted turkey were studied by testing the initial inhibition as well as the inhibition during storage. The initial antimicrobial effects of WPI coatings incorporating LPOS (LPOS-WPI coatings) were examined with various inoculation levels and LPOS concentrations. LPOS-WPI coatings with 7 and 4% of LPOS demonstrated initial 3- and 2-log CFU/g reductions of S. enterica and E. coli O157:H7, respectively. The antimicrobial effect was observed regardless of whether the turkey was inoculated before or after coating. Storage studies were conducted for 42 days at 4 and 10 degrees C with S. enterica (6.0 log CFU/g)- or E. coli O157:H7 (5.6 log CFU/g)-inoculated sliced turkey. LPOS concentrations for the storage studies of S. enterica and E. coli O157:H7 were 5 and 3% (wt/wt), respectively, in the coating solution and in an LPOS solution for spreading. LPOS-WPI coatings inhibited the growth of both S. enterica and E. coli O157:H7 in turkey at both 4 and 10 degrees C for 42 days. The inhibition was more pronounced when the coating was formed on the surface of the turkey prior to inoculation than when the coating was formed on the inoculated surface. More effective inhibition of S. enterica and E. coli O157:H7 was observed with the LPOS-WPI coatings than with the LPOS solution-spreading treatment. LPOS-WPI coatings also retarded the growth of total aerobes during storage.

The sensitisation of Escherichia coli towards lysozyme, nisin and lactoperoxidase by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied. At above 150 MPa, E. coli became sensitive to lysozyme and nisin when these compounds were added before the HPH treatment, but cells treated by HPH remained insensitive for lysozyme and nisin added after that treatment. No sensitisation to the lactoperoxidase enzyme system was observed, and no evidence of cytoplasmic membrane damage could be found in cells surviving HPH treatment using SYTO 9/propidium iodide staining. A previously isolated spontaneous E. coli mutant that is resistant to lysozyme under high hydrostatic pressure, was also found to be resistant to lysozyme upon HPH treatment. Further, lysozyme resistance in this mutant was not due to enhanced levels of periplasmic lysozyme inhibitor (Ivy), since knock-out of the ivy gene did not increase the sensitivity of the mutant to lysozyme upon HPH treatment. These results indicate that HPH treatment at pressures up to 300 MPa, unlike treatment with high hydrostatic pressure, does not cause sublethal injury, and thus does not sensitise bacteria to antimicrobial compounds that disturb cellular homeostasis such as might be the case for the lactoperoxidase system. However, like high hydrostatic pressure treatment, HPH may sensitise E. coli to lysozyme and nisin by inducing a transient permeabilisation of the outer membrane that does not involve a physical disruption and that is immediately repaired after the process.