The Manufacturing and Characterisation of Eugenol-Enclosed Liposomes Produced by Microfluidic Method.

In this study, liposomes enclosing eugenol were prepared using microfluidics. Two lipids-1,2-dimyristoyl-sn-glycero-3-phosphocholine, 18:0 (DSPC) and 2-dimyristoyl-sn-glycero-3-phosphocholine, 14:0 (DMPC)-and microfluidic chips with serpentine and Y-shaped micromixing designs were used for the liposomal formulation. Minimum bactericidal concentration (MBC) values indicated that eugenol was more effective against Gram-negative than Gram-positive bacteria. Four different flow-rate ratios (FRR 2:1, 3:1, 4:1, 5:1) were explored. All liposomes' encapsulation efficiency (EE) was determined: 94.34% for DSPC 3:1 and 78.63% for DMPC 5:1. The highest eugenol release of 99.86% was observed at pH 4, DMPC 3:1 (Y-shaped chip). Liposomes were physically stable at 4, 20 and 37 °C for 60 days as determined by their size, polydispersity index (PDI) and zeta potential (ZP). The most stable liposomes were observed at FRR 5:1 for DSPC. EE, stability, and eugenol release studies proved that the liposomal formulations produced can be used as delivery vehicles to increase food safety.

Development of Geraniol-Loaded Liposomal Nanoformulations against Salmonella Colonization in the Pig Gut.

Salmonella is a global health threat, with pig production being one of the main sources of human salmonellosis. The current study investigated the antivirulence properties of geraniol for inhibiting the in vitro colonization of Salmonella. The minimum inhibitory (MIC) and bactericidal concentrations (MBC) of geraniol against Salmonella typhimurium followed by the sub-MIC of geraniol were determined. Results provided clear evidence that geraniol at 1/8 MIC can be used as an effective, non-toxic antivirulence compound to inhibit virulence factors (motility, adhesion, and invasiveness) affecting the colonization of S. typhimurium on IPEC-J2 cells. Additionally, the findings signified that microfluidics is an emerging technology suitable for the preparation of stable liposomes with a small size (<200 nm) and high encapsulation efficiency (EE) of up to 92.53%, which can act as effective carriers of geraniol into the pig gastrointestinal tract (GIT), targeting Salmonella, preventing colonization, and thus increasing the safety of the food supply chain.

Inhibition of corrosion causing Pseudomonas aeruginosa using plasma-activated water.

AIMS: The cost of Microbiologically Influenced Corrosion (MIC) significantly affects a wide range of sectors. This study aims to assess the efficiency of a novel technology based on the use of plasma-activated water (PAW) in inhibiting corrosion caused by bacteria. METHODS AND RESULTS: This study evaluated the effectiveness of PAW, produced by a plasma bubble reactor, in reducing corrosion causing Pseudomonas aeruginosa planktonic cells in tap water and biofilms were grown onto stainless steel (SS) coupons. Planktonic cells and biofilms were treated with PAW at different discharge frequencies (500-1500 Hz) and exposure times (0-20 min). P. aeruginosa cells in tap water were significantly reduced after treatment, with higher exposure times and discharge frequencies achieving higher reductions. Also, PAW treatment led to a gradual reduction for young and mature biofilms, achieving >4-Log reductions after 20 min. Results were also used to develop two predictive inactivation models. CONCLUSIONS: This work presents evidence that PAW can be used to inactivate both planktonic cells and biofilms of P. aeruginosa. Experimental and theoretical results also demonstrate that reduction is dependent on discharge frequency and exposure time. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the potential of using PAW as means to control MIC.

An Electrocatalytic Screen-Printed Amperometric Sensor for the Selective Measurement of Thiamine (Vitamin B1) in Food Supplements.

An electrocatalytic screen-printed sensor has been investigated for the measurement of the biologically important biomolecule vitamin B1 (thiamine) for the first time in food supplements. Under basic conditions, the vitamin was converted to its electrochemically active thiolate anion species. It was shown that an electrocatalytic oxidation reaction occurred with the screen-printed carbon electrode containing the mediator cobalt phthalocyanine (CoPC-SPCE). This had the advantage of producing an analytical response current at an operating potential of 0 V vs. Ag/AgCl compared to +0.34 V obtained with plain SPCEs. This resulted in improved selectivity and limit of detection. Detailed studies on the underlying mechanism occurring with the sensor are reported in this paper. A linear response was obtained between 0.1 and 20 µg mL-1, which was suitable for the quantification of the vitamin in two commercial products containing vitamin B1. The mean recovery for a multivitamin tablet with a declared content of 5 mg was 101% (coefficient of variation (CV) of 9.6%). A multivitamin drink, which had a much lower concentration of vitamin B1 (0.22 mg/100 mL), gave a mean recovery of 93.3% (CV 7.2%). These results indicate that our sensor holds promise for quality control of food supplements and other food types.

7-Hydroxylation of warfarin is strongly inhibited by sesamin, but not by episesamin, caffeic and ferulic acids in human hepatic microsomes.

Warfarin is a commonly used anticoagulant drug and is a derivate of coumarin. Cytochrome P450 2C9 (CYP2C9) plays the key role in transformation of coumarin and thus, influences determination of warfarin dosage. A number of factors including dietary compounds such as sesamin, caffeic acid and ferulic acids can regulate the activity of CYP2C9. The present study tested the hypothesis that sesamin, episesamin, caffeic acid and ferulic acid decreases the rate of warfarin 7-hydroxylation via inhibition of hepatic CYP2C9. The experiments were conducted on hepatic microsomes from human donors. It was demonstrated that the rate of 7-hydroxylation of warfarin was significantly decreased in the presence of sesamin in the range of concentrations from 5 to 500 nM, and was not affected by episesamin, caffeic acid and ferulic acid in the same range of concentrations. The kinetic analysis indicated non-competitive type of inhibition by sesamin with Ki = 202 ±â€¯18 nM. In conclusion, the results of our in vitro study revealed that sesamin was able to inhibit formation of a major metabolite of warfarin, 7-hydroxywarfarin. The potentially negative consequences of the consumption of high amounts of sesamin-containing food or dietary supplements in warfarin-treated patients need to be further studied.

Polymorphisms of the porcine cathepsins, growth hormone-releasing hormone and leptin receptor genes and their association with meat quality traits in Ukrainian Large White breed.

Cathepsins, growth hormone-releasing hormone (GHRH) and leptin receptor (LEPR) genes have been receiving increasing attention as potential markers for meat quality and pig performance traits. This study investigated the allele variants in four cathepsin genes (CTSB, CTSK, CTSL, CTSS), GHRH and LEPR in pure-bred Ukrainian Large White pigs and evaluated effects of the allele variants on meat quality characteristics. The study was conducted on 72 pigs. Genotyping was performed using PCR-RFLP technique. Meat quality characteristics analysed were intramuscular fat content, tenderness, total water content, ultimate pH, crude protein and ashes. A medium level of heterozygosity values was established for GHRH and LEPR genes which corresponded to very high levels of informativeness indexes. Cathepsins CTSL, CTSB and CTSK had a low level of heterozygosity, and CTSS did not segregate in this breed. Association studies established that intramuscular fat content and tenderness were affected by the allele variance in GHRH and LEPR but not by CTSB and CTSL genes. The GHRH results could be particularly relevant for the production of lean prime cuts as the A allele is associated with both, a lower meat fat content and better tenderness values, which are two attributes highly regarded by consumers. Results of this study suggest that selective breeding towards GHRH/AA genotype would be particularly useful for improving meat quality characteristics in the production systems involving lean Large White lines, which typically have less than 2 % intramuscular fat content.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs.

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

Metabolism of androstenone, 17ß-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3ß-hydroxysteroid dehydrogenase in this process.

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17ß-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3ßHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3ßHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3ß-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17ß-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3ßHSD enzyme was not involved in 17ß-estradiol metabolism. 5α-Dihydrotestosterone-17ß-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3ßHSD enzyme activity as demonstrated by inhibition study.

Combined effects of dietary arginine, leucine and protein levels on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of crossbred pigs.

The cumulative effects of dietary arginine, leucine and protein levels on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig longissimus lumborum muscle and subcutaneous adipose tissue (SAT) were investigated. The experiment was performed on fifty-four intact male pigs (Duroc × Pietrain × Large White × Landrace crossbred), with a live weight ranging from 59 to 92 kg. The pigs were randomly assigned to one of six experimental treatments (n 9). The treatments followed a 2 × 3 factorial arrangement, with two levels of arginine supplementation (0 v. 1 %) and three levels of a basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet to achieve 2 % of leucine, RPDL). The results showed that dietary arginine supplementation did not affect the intramuscular fat (IMF) content and back fat thickness, but increased the total fat in SAT. This effect was associated with an increase in fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) mRNA levels in SAT, which suggests that arginine might be involved in the differential regulation of some key lipogenic genes in pig muscle and SAT. The increase in IMF content under the RPD, with or without leucine supplementation, was accompanied by increased FASN and SCD mRNA levels. Arginine supplementation did not influence the percentage of main fatty acids, while the RPD had a significant effect on fatty acid composition in both tissues. Leucine supplementation of RPD did not change IMF, total fat of SAT and back fat thickness, but increased 16 : 0 and 18 : 1cis-9 and decreased 18 : 2n-6 in muscle.

Effect of breed on fatty acid composition and lipogenic enzyme abundance in the subcutaneous adipose tissue of pigs.

UNLABELLED: This study investigated the role of lipogenic enzyme expression in breed-specific fat deposition in pigs. OBJECTIVES: (i) determine effect of breed on the relative abundance of the key lipogenic enzymes stearoyl-CoA desaturase (SCD), delta-6 desaturase (Δ6D), and fatty acid synthase (FAS) in pig subcutaneous adipose tissue. (ii) Investigate breed-specific relationships between lipogenic enzyme abundance and fatty acid composition. Large White × Piétrain, Piétrain, and Duroc × Piétrain pigs were used. Expression of SCD, Δ6D, and FAS was analyzed by Western blotting. Fatty acid composition was determined by gas chromatography. FAS protein in Large White × Piétrain pigs was similar to the Piétrain breed, but was significantly higher than Duroc × Piétrain. A positive relationship was found between FAS abundance and the saturated fatty acids (SFAs), for Large White × Piétrain pigs, but not for the other breeds. Δ6D was significantly higher in Large White × Piétrain compared with Duroc × Piétrain and Piétrain. This was accompanied by significantly higher total n-3 poly-unsaturated fatty acids (PUFAs) in the Large White × Piétrain when compared to the other breeds. CONCLUSIONS: (i) increased subcutaneous adipose tissue SFA content in Large White × Piétrain pigs (but not Piétrain and Duroc × Piétrain) is related to increased abundance of FAS protein; (ii) high n-3 PUFA content in Large White × Piétrain pigs is related to activation of Δ6D protein synthesis; (iii) SCD and Δ6D abundance does not contribute to between-breed differences in MUFA and n-6 PUFA content of pig subcutaneous adipose tissue.

TRPM8 agonists modulate contraction of the pig urinary bladder.

The transient receptor potential melastin-8 (TRPM8) channel is activated by the "cooling" compounds menthol and icilin. Pathophysiologically, it is implicated in the overactive bladder and bladder cooling reflex, but the activity of TRPM8 in normal bladder physiology is poorly understood. We investigated the distribution of TRPM8 channels and the effect of TRPM8 agonists on the contractile function of pig bladder (n = 35) strips and whole bladders. The distribution of TRPM8 was examined by immunohistochemistry. The effect of vesical or intravascular menthol (0.1-0.3 mmol/L) or icilin (50 µmol/L) on carbachol-induced isolated whole bladder contractions was monitored by recording vesical pressure. Strips of denuded detrusor or mucosa were mounted in organ baths to study the effect of TRPM8 agonists on the contractile responses to 10 µmol/L carbachol. TRPM8-like immunoreactivity was detected on pig urothelium. Intravascular menthol (0.3 mmol/L) and icilin (50 µmol/L) significantly decreased the magnitude of carbachol-induced whole bladder contraction, whereas vesical administration significantly increased the response. In detrusor and mucosal strips, both menthol (0.3 mmol/L) and icilin (50 µmol/L) inhibited carbachol-induced contractions. We conclude that the TRPM8 channel is expressed on the urothelium of pig bladder. In the whole organ, exposure of the urothelium to menthol or icilin increases the contractile response to carbachol. Where detrusor muscle is exposed directly to these compounds, the contractile response to carbachol is reduced.

Use of whole blood for analysis of disease-associated biomarkers.

Analyses for diagnosis and monitoring of pathological conditions often rely on blood samples, partly due to relative ease of collection. However, many interfering substances largely preclude the use of whole blood itself, necessitating separation of plasma or serum. We present a feasibility study demonstrating potential use of fresh or frozen whole blood to detect soluble biomarkers using an enzyme-linked immunosorbent assay (ELISA)-based method. Good correlation between levels of soluble CD25 in plasma and whole blood of healthy individuals or Alzheimer's patients was established. These results provide a basis for development of a novel biosensor approach for disease-associated biomarker detection in whole blood.

Differential effects of reduced protein diets on fatty acid composition and gene expression in muscle and subcutaneous adipose tissue of Alentejana purebred and Large White × Landrace × Pietrain crossbred pigs.

The present study assessed the effect of pig genotype (fatty v. lean) and dietary protein and lysine (Lys) levels (normal v. reduced) on intramuscular fat (IMF) content, subcutaneous adipose tissue (SAT) deposition, fatty acid composition and mRNA levels of genes controlling lipid metabolism. The experiment was conducted on sixty intact male pigs (thirty Alentejana purebred and thirty Large White × Landrace × Pietrain crossbred), from 60 to 93 kg of live weight. Animals were divided into three groups fed with the following diets: control diet equilibrated for Lys (17·5 % crude protein (CP) and 0·7 % Lys), reduced protein diet (RPD) equilibrated for Lys (13·2 % CP and 0·6 % Lys) and RPD not equilibrated for Lys (13·1 % CP and 0·4 % Lys). It was shown that the RPD increased fat deposition in the longissimus lumborum muscle in the lean but not in the fatty pig genotype. It is strongly suggested that the effect of RPD on the longissimus lumborum muscle of crossbred pigs is mediated via Lys restriction. The increase in IMF content under the RPD was accompanied by increased stearoyl-CoA desaturase (SCD) and PPARG mRNA levels. RPD did not alter backfat thickness, but increased the total fatty acid content in both lean and fatty pig genotype. The higher amount of SAT in fatty pigs, when compared with the lean ones, was associated with the higher expression levels of ACACA, CEBPA, FASN and SCD genes. Taken together, the data indicate that the mechanisms regulating fat deposition in pigs are genotype and tissue specific, and are associated with the expression regulation of the key lipogenic genes.

Investigation of testosterone, androstenone, and estradiol metabolism in HepG2 cells and primary culture pig hepatocytes and their effects on 17ßHSD7 gene expression.

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17ß-hydroxysteroid dehydrogenase type 7 (17ßHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17ßHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17ßHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17ßHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3'-hydroxy compound 3ß-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for ß-estradiol. Inhibition study with 17ßHSD inhibitor apigenin showed that apigenin didn't affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and ß-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17ßHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.

Δ(9) desaturase protein expression and fatty acid composition of longissimus dorsi muscle in lambs fed green herbage or concentrate with or without added tannins.

The aims of this study were to investigate the effect of feeding system and of supplementation of tannins (8.93% DM) on the relationship between intramuscular fat content, fatty acid composition and Δ(9)desaturase (Δ(9)d) protein expression in longissimus dorsi muscle of lamb. Twenty-eight Comisana lambs (age 45days) were fed either vetch (Vicia sativa) or concentrate. The herbage diet was (i) lower in saturated fatty acids (especially in C16:0), C18:1 n-9 and in C18:2 n-6; (ii) higher in C16:1 and C18:3 n-3 when compared to concentrate. Within each feeding system the lambs were divided into two sub-groups, one of which received the diet without tannins supplementation, and the other was fed the diets supplemented with the tannins from Quebracho (Schinopsis lorentzii). The animals were slaughtered at age 105days. The concentrate feeding system increased (p<0.01) the total intramuscular fat content and the amount of SFA, MUFA and n-6 PUFA and decreased the level of n-3 PUFA (p=0.05) when compared to the vetch-fed animals but did not affect Δ(9) desaturase protein expression. There was no correlation between Δ(9)d protein expression and total intramuscular fatty acids, CLA and MUFA level. It was suggested that in ruminants, in contrast to monogastric animals, Δ(9)d expression does not play the key role in intramuscular fatty acids formation. Tannins supplementation resulted in higher (p<0.05) muscle levels of transC18:1 and C18:2 n-6. It has also increased Δ(9)d expression in the case of herbage-based diet but not in the case of concentrate-based diet. The mechanism of tannins action on the enzyme expression needs to be elucidated.

Regulation of CYP2A6 protein expression by skatole, indole, and testicular steroids in primary cultured pig hepatocytes.

CYP2A6 is one of the enzymes involved in the hepatic metabolism of a naturally produced compound, skatole, in the pig. Low CYP2A6 activity has been linked to excessive accumulation of skatole in pig adipose tissue and development of the phenomenon "boar taint." CYP2A6 activity varies between male and female animals, suggesting the involvement of sex hormones in regulation of the enzyme activity and/or expression. The present study investigated whether pig hepatic CYP2A6 protein expression is regulated by the testicular steroids testosterone, androstenone, or estrone sulfate using primary cultured hepatocytes as a model system. The study has also examined whether CYP2A6 expression can be modulated by the boar taint compounds skatole and indole. The research has established that androstenone inhibits CYP2A6 protein expression at the concentration of 1, 10, and 100 nM by 55, 37, and 44%, respectively. In contrast to androstenone, skatole and indole (final concentrations, 1, 10, and 100 nM) had a stimulatory effect on CYP2A6 expression. The effect of indole was more pronounced than that of skatole (maximum induction by 145 and 70%, respectively). Estrone sulfate and testosterone did not have a significant effect on CYP2A6 protein level. This is, as far as we know, the first communication to report the regulation of pig hepatic CYP2A6 expression by steroids and boar taint compounds. The hormonal modulation of CYP2A6 expression might contribute to gender-related differences in pig hepatic CYP2A6 activity and skatole accumulation in pig adipose tissue.