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.

Flaminal® versus Flamazine® in the treatment of partial thickness burns: A randomized controlled trial on clinical effectiveness and scar quality (FLAM study).

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.

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.

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.

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.

Two novel roles of buffalo milk lactoperoxidase, antibiofilm agent and immunomodulator against multidrug resistant Salmonella enterica serovar Typhi and Listeria monocytogenes.

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.

Effects of lactoferrin and lactoperoxidase-containing food on the oral microbiota of older individuals.

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.

Effectiveness of casein phosphopeptide-amorphous calcium phosphate and lysozyme, lactoferrin, and lactoperoxidase in reducing Streptococcus mutans counts in dentinal caries.

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.

Short communication: The influence of solids concentration and bleaching agent on bleaching efficacy and flavor of sweet whey powder.

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.

Chitosan coating incorporated with the lactoperoxidase system: an active edible coating for fish preservation.

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.

Exploring the potential anti-thyroid activity of Acetyl-L-carnitine: Lactoperoxidase inhibition profile, iodine complexation and scavenging power against H2O2. Experimental and theoretical studies.

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 (Kc = 8.07 ± 0.32 x 105 M-1), inhibit the enzyme lactoperoxidase (LPO) (IC50 = 17.60 ± 0.76 µM), and scavenge H2O2 (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.

Cold enzymatic bleaching of fluid whey.

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.

Milk-derived proteins and peptides in clinical trials.

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

Inhibition of biofilms by glucose oxidase, lactoperoxidase and guaiacol: the active antibacterial component in an enzyme alginogel.

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.

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

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

The use of lactoperoxidase for the bleaching of fluid whey.

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.

Augmenting apoptosis-mediated anticancer activity of lactoperoxidase and lactoferrin by nanocombination with copper and iron hybrid nanometals.

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 (IC50) 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.

The Impact of Mouthwash on the Oropharyngeal Microbiota of Men Who Have Sex with Men: a Substudy of the OMEGA Trial.

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.

Influences of hyaluronic acid on the anticandidal activities of lysozyme and the peroxidase system.

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.

Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis.

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.