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Relationship between Molecular Structure Characteristics of Feed Proteins and Protein In vitro Digestibility and Solubility

The nutritional value of feed proteins and their utilization by livestock are related not only to the chemical composition but also to the structure of feed proteins, but few studies thus far have investigated the relationship between the structure of feed proteins and their solubility as well as digestibility in monogastric animals. To address this question we analyzed soybean meal, fish meal, corn distiller’s dried grains with solubles, corn gluten meal, and feather meal by Fourier transform infrared (FTIR) spectroscopy to determine the protein molecular spectral band characteristics for amides I and II as well as α-helices and β-sheets and their ratios. Protein solubility and in vitro digestibility were measured with the Kjeldahl method using 0.2% KOH solution and the pepsin-pancreatin two-step enzymatic method, respectively. We found that all measured spectral band intensities (height and area) of feed proteins were correlated with their the in vitro digestibility and solubility (p≤0.003); moreover, the relatively quantitative amounts of α-helices, random coils, and α-helix to β-sheet ratio in protein secondary structures were positively correlated with protein in vitro digestibility and solubility (p≤0.004). On the other hand, the percentage of β-sheet structures was negatively correlated with protein in vitro digestibility (p<0.001) and solubility (p = 0.002). These results demonstrate that the molecular structure characteristics of feed proteins are closely related to their in vitro digestibility at 28 h and solubility. Furthermore, the α-helix-to-β-sheet ratio can be used to predict the nutritional value of feed proteins.

INTRODUCTION
Nutritional value of protein in feedstuff not only depends on its amino acid content and composition proportion, but also on its molecular structure characteristics (Peng et al., 2014). Recent studies have highlighted the importance of determining the relationship between molecular spectral band characteristics of proteins and their intestinal absorption so as to clarify the mechanism of nitrogen supplementation (Qin et al., 2014).
The traditional method of determining the nutritional value of feeds involves destroying the three-dimensional structure of constituent proteins by acid or alkali treatment or by mechanical polishing, and then using so-called wet chemical methods to determine chemical composition. However, this approach provides no information at a molecular level on the nutritional value of feeds or on their capacity for digestion by livestock. Fourier transform infrared (FTIR) technique is a powerful tool for analyzing the structure of feed proteins (Xin et al., 2013a), which allows investigators to predict how they are processed and absorbed within the animals' digestive tract. However, there is certainly difficulty to use FTIR spectrum for quantifying the tertiary structure having higher portion of side chains of the conventional feedstuffs protein. al., 2013). Specifically, α-helices and β-sheets in the protein secondary structures can affect digestibility of feed proteins (Dyson and Wright, 1993). Certain features (amide I and II bands) of protein molecular structures of corn distiller's dried grains with solubles (corn DDGS) from various sources were correlated with intestinal protein digestibility, which can be used to predict the absorption of essential nutrients by the small intestine (Chen et al., 2014). Another study found that spectral band intensity characteristics of proteins affect protein quality, nutrient utilization, and digestion in dairy cows (Doiron et al., 2009). Therefore, establishing a relationship between the molecular structure characteristics of conventional feed proteins and their nutritional value will strengthen the current knowledge on protein nutritional value and broaden the scope of molecular spectroscopy as a bioanalytical tool for rapidly assessing the nutritive value of feed proteins.
To our knowledge, the relationship between molecular structure characteristics of conventional feed proteins and protein solubility (PS) and in vitro digestibility in monogastric animals has not been previously investigated. The objectives of this study were i) to assess protein molecular structure characteristics of conventional feed proteins by FTIR; ii) to examine the PS and in vitro digestibility of these proteins; and iii) to quantify the relationship between protein molecular structure characteristics and PS as well as in vitro digestibility.

Sample preparation and treatment
Five conventional feed ingredients (soybean meal, fish meal, corn DDGS, corn gluten meal, and feather meal) were provided by Changchun Wellhope Animal Husbandry Co. (Changchun, China). All feed samples used in this experiment were collected from raw commodities and stored according to commodity storage standard, one month of storage period. One half of each sample was ground in the laboratory mill through a 1 mm screen for laboratory analysis and sub-samples (n = 3 per type of feed) was collected and stored at 4°C. The other half was stored as a backup at -20°C. Chemical profiles of the five feeds are presented in Table 1.

Determination of protein solubility
PS was determined according to the method as described by Dale (1987). Feedstuff sample with 1.5 g was placed in a 100 mL conical flask, and then added 75 mL of 0.2% (w/v) potassium hydroxide solution; the mixture was stirred on a magnetic stirrer for 20 min and then transferred to a 50 mL tube and centrifuged at 2,700 rpm for 10 min. 15 mL volume of supernatant was measured by the chemical method (AOAC, 1990), yielding an amount equivalent to 0.3 g of original sample (15/75 = x/1.5 g, where x = 0.3 g). PS (%) was calculated as crude protein (CP) content in 15 mL supernatant/CP content of the original sample×100%.

Determination of protein in vitro digestibility
Protein in vitro digestibility was evaluated according to a method as previously described by Boisen and Fernandez (1995). Feedstuff sample with 1.0 g (accurate to 0.001 g) was placed in a 100 mL conical flask, and then added 10 mL of 0.01 M hydrochloric acid (pH 2.0) and pepsin solution containing 1.0 mg porcine pepsin (product no. P7-000; Sigma, St. Louis, MO, USA). To prevent bacterial growth, 0.5 mL of chloramphenicol solution consisting of 0.5 g chloramphenicol (product no. 0230; Amresco, Solon, OH, USA) in 100 mL ethanol was added to the mixture, followed by incubation for 4 h at 37℃. After neutralization with 0.2 M sodium hydroxide, 10 mL of 0.2 M phosphate buffer (pH 6.8) was added. The pH was adjusted to 6.8 with 1 M HCl or 1 M NaOH, and the solution was mixed with 1 mL of a freshly prepared pancreatin solution containing 50 mg porcine pancreatin (product no.P7-545-100G; Sigma, USA). The flask was closed with a rubber stopper and incubated with continuous magnetic stirring at 39°C for 4, 8, 12, 16, 20, 24, and 28 h, respectively. After adding 5 mL of 20% sulfosalicylic acid, the sample was centrifuged at 15,000 rpm for 15 min; the supernatant was discarded and the precipitate was heated at 80°C for 24 h. CP digestibility (in %) was calculated as CP content of the original sample −CP content of precipitate/CP content of the original sample×100%.

Fourier transform infrared spectroscopy
FTIR spectroscopy was performed according to the method as described by Long et al (2015). Briefly, 2 mg of sample was mixed with 200 mg KBr in a vacuum dryer for 24 h; the mixture was uniformly ground and pressed into a tablet (tablet thickness = 0.25 mm; translucent shape) using an infrared (IR) tablet press (FW-4; Thermo Fisher Scientific, Waltham, MA, USA). FTIR spectra (FTIR-8400s; Shimadzu, Kyoto, Japan) were acquired in the mid-IR range (4,000 to 400 cm −1 ) at a resolution of 4 cm −1 with 64 co-added scans. Each sample was run six times. As a control, a KBr pellet without protein was recorded under identical conditions. IR spectra were processed using OMNIC 8.0 software (Nicolet Analytical Instruments, Madison, WI, USA), and after baseline correction of the original map the sample and control spectra were compared. Amide I band narrowing was achieved with a full height of 38.2 and a resolution enhancement factor of 4.4. Fourier self-deconvolution using Origin 7.5 software (Origin Lab, Northampton, MA, USA) was used for Gaussian curve fitting in the region of the amide I band to separate overlapping bands. The secondary structure content of samples was detected from IR second-derivative amide I spectra by manually computing the relative peak areas under the bands assigned to a particular substructure.

Statistical analysis
Data were evaluated by analysis of variance with SPSS v.19.0.1 software (SPSS Inc., Chicago, IL, USA). The model used for protein molecular spectral band characteristics, PS and in vitro digestibility analysis was Y ij = μ i +e ij , where Y ij is an observation on the dependent variable ij (i = 1, 2,. . .,5; j = 1, 2, 3), μ i is the overall mean (i = 1, 2,. . .,5), and e ij is the random error associated with the observation ij. Multiple comparisons of group means were carried out using Duncan's method. Results are expressed as mean±standard error, with α = 0.05 indicating a statistically significant difference. Correlation analysis was performed using Pearson's correlation coefficient with 95% confidence limits (p<0.05).

Protein spectral band intensities
The protein IR spectrum showed two salient features, i.e., the amide I (1,700 to 1,600 cm -1 ) and amide II (1,600 to 1,500 cm -1 ) bands (Figure 1). Results of the protein spectral band intensities analyses are presented in Table 2. Significant differences were observed among feeds in terms of amide II heights and areas and amide I-to-II height and area ratios (p<0.001). Soybean meal had the highest values for amide I and II heights, amide I and II areas, and amide I-to-II height and area ratios, whereas corn DDGS had the lowest values for all of these features.

Protein secondary structures and solubility
The Gaussian curve fitting analysis of the amide I band peak was carried out based on the preliminary study of Meng-Xia and Yuan (2002), showed characteristic peaks at 1,611 to 1,639 cm -1 that were attributed to β-sheets. Other peaks were observed that corresponded to the following structures: random coils, 1,640 to 1,649 cm -1 ; α-helices, 1,650 to 1,658 cm -1 ; and β-turns, 1,660 to 1,700 cm -1 (Figure 2). Results of the protein secondary structures and solubility analyses are shown in Table 3. Soybean meal had the highest percentage of α-helices and random coil structures and α-helix-to-β-sheet ratios; feather meal had the highest percentage of β-sheet structures; and corn DDGS had the highest percentage of β-turn structures, while corn gluten meal had the lowest percentage of β-turn Wavenumbers are as follows: amide I height, 1,646 cm -1 ; amide II height, 1,542 cm -1 ; amide I area, 1,720 to 1,583 cm -1 ; and amide II area, 1,583 to 1,479 cm -1 .   and random coil structures. The rank order of PS was soybean meal>fish meal>corn DDGS>corn gluten meal>feather meal (p<0.05).

Protein in vitro digestibility
Evaluation of protein in vitro digestibility revealed significant differences among five conventional feed ingredients at each time point (p<0.05; Figure 3). The in vitro digestibility of proteins by pepsin-trysin increased over time. Soybean and feather meal had the highest and lowest in vitro digestibility values, respectively, at 28 h. The rank order of protein in vitro digestibility at this time point was soybean meal>fish meal>corn DDGS>corn gluten meal>feather meal (p<0.05). The lower in vitro digestibility of corn DDGS proteins at 28 h as compared to that at 24 h may be explained by the inhibition of enzymatic reactions due to accumulation of digestion products.

Correlation between protein spectral band intensities and protein in vitro digestibility and solubility
Coefficients of correlation between protein spectral band intensities (height and area) and protein in vitro digestibility at 28 h as well as solubility are shown in Table  4. Amide I height was positively correlated with PS (r = 0.958, p<0.001) and in vitro digestibility (r = 0.848, p< 0.001), as were amide II height (r = 0.707, p = 0.003 and r = 0.518, p = 0.048, respectively), the height ratio of amide I to II (r = 0.857, p<0.001 and r = 0.894, p<0.001, respectively), amide I area (r = 0.959, p<0.001 and r = 0.959, p<0.001, respectively), amide II area (r = 0.709, p = 0.003 and r = 0.519, p = 0.047, respectively), and area ratio of amide I to II (r = 0.854, p<0.001 and r = 0.893, p<0.001, respectively).

Correlation between protein secondary structures and protein in vitro digestibility and solubility
Coefficients of correlation between protein secondary structures and protein in vitro digestibility at 28 h as well as solubility are shown in Table 5. The percentage of α-helix structures was positively correlated with PS (r = 0.903, p<0.001) and in vitro digestibility (r = 0.916, p<0.001), as were the percentage of random coil structures (r = 0.694, p   = 0.004 and r = 0.789, p<0.001, respectively) and α-helixto-β-sheet ratios (r = 0.955, p<0.001 and r = 0.966, p<0.001, respectively). However, the percentage of β-sheet structures was negatively correlated with both protein in vitro digestibility (r = -0.826, p<0.001) and solubility (r = -0.740, p = 0.002).

DISCUSSION
There is an increasing demand to improve the nutritional value of feed proteins for monogastric animals by optimizing feed protein sources and increasing the efficiency of protein utilization, which requires a fast and accurate estimation of the intestinal digestibility of feed proteins. Earlier findings suggest that the pepsin-pancreatin two-step enzymatic method is potentially a useful for evaluating in vivo digestibility (Boisen et al., 1995). However, previous studies have been unable to explain the variability in the digestion of different feed proteins, which are likely due to differences in protein structure.
The present study investigated the molecular spectral band characteristics of proteins in various types of feedstuff in relation to their digestibility and solubility. PS and protein spectral band intensities (height and area) in the feedstuff examined in this study varied widely. Protein secondary structures such as α-helices, β-sheets, and random coils as well as α-helix-to-β-sheet ratios in soybean meal and corn DDGS, and the relative quantitative amounts of α-helices and β-sheets in feather meal were inconsistent with earlier findings (Yu et al., 2004;Jiao et al., 2012). Even the reason for these differences is unclear, they may be related to variations inherent in feed sources, processing technology, or storage conditions (Peng et al., 2014).
We found that in vitro digestibility of soybean meal protein was lower than previously reported values (Boisen et al., 1995), which may have been due to the presence of anti-nutritional factors (Tan-Wilson et al., 1985) that can inhibit enzymatic reactions. On the other hand, fish meal protein in vitro digestibility was higher than that reported in another study (Huang et al., 2000) whereas feather meal protein in vitro digestibility was similar to results obtained by other investigators (Yu et al., 2004). This may be due to the presence of keratin-a scleroprotein that makes up 85% to 90% of feather meal-which has highly stable S-S and S-H linkages that are not readily broken down by animals without processing (Papadopoulos et al., 1986). One possible reason for which corn DDGS and gluten meal had lower protein in vivo digestibility and PS is their low solubility in water due to a high content of alcohol-soluble proteins containing hydrophobic amino acids (Liu et al., 2013); alternatively, the S-S or O-H bonds in their structures may promote the formation of α-helices (Zhang and Yu, 2012), which are structurally stable and therefore not easily digested.
The amide I band mainly arises from stretching vibrations of the amide C=O group, while the amide II band is attributable to N-H bending (60%) and C-N stretching (40%) vibrations; these have also been used to determine protein structure or conformation (Yan et al., 2014), and the protein two primary spectral band intensities in terms of peak height and peak area indicate quantitative differences in protein functional groups (Peng et al., 2014). Corn DDGS and feather meal degraded slowly as compared to the other types of feed due in part to their low basic amino acid content, which is indirectly reflected by amide height and area. Trypsin is the main protease in the intestine and targets the peptide bond between lysine and arginine; therefore, the lack of basic amino acids would diminish the probability of contact between the CP in feed and digestive enzymes in the animal, thereby decreasing the amount and variety of oligopeptides and free amino acids that are released (Adler-Nissen, 1979).
The vibrational frequency of the amide I band is particularly sensitive to and can be used to predict protein secondary structure (Yu et al., 2004). Our findings confirmed that the percentage of β-sheet structures is closely related to the nutritional value of feed, with higher percentage being associated with lower PS and CP digestibility in the small intestine, since β-sheets have a large number of hydrogen bonds that can hinder protease activity. We also confirmed that the nutritional value of proteins feed differed according to α-helix-to-β-sheet ratio; previous studies have also reported that this ratio is a good predictor of nutritional value or digestibility of proteins feed Xin et al., 2013b). We found that the percentage of random coil structures was positively correlated with PS and digestibility, possibly because this structure is strong and flexible and has fewer hydrogen bonds that can impede enzyme access, which allows proteins to be readily degraded.


extfieldsofstudy: Chemistry Medicine

provenance: AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2015.jsonl.gz.gz

text:
Comparing the effect of encapsulated and unencapsulated fennel extracts on the shelf life of minced common kilka (Clupeonella cultriventris caspia) and Pseudomonas aeruginosa inoculated in the mince

Abstract The quality of minced kilka (Clupeonella cultriventris caspia) with gum arabic encapsulated (0.3% and 0.5% w/w) and unencapsulated fennel extract (FE) (0.3% and 0.5% w/w) stored at 4°C was examined over a storage period of 15 days. The control and the treated fish samples were analyzed periodically for microbiological (total viable count [TVC] and total psychrotrophic count [TPC]) and chemical (peroxide value (PV) and total volatile nitrogen (TVB‐N)) parameters. Also the inhibitory effect of encapsulated and unencapsulated FE was evaluated against Pseudomonas aeruginosa, inoculated in minced kilka. According to the results, encapsulated FE samples showed the lowest amount of lipid oxidation and microbial deterioration during the storage period compared with the control and pure extract treatments. Although, the encapsulated FE at 0.5% showed drastic bacterial effect against Pseudomonas aeruginosa compared to others. Generally, gum arabic encapsulation could help to obtain higher antimicrobial and antioxidant activity in lower FE concentrations in minced fish.

Introduction
Kilka is the most abundant fish in the Caspian Sea located in the north of Iran. Three species of Kilka are anchovy Kilka (Clupeonella engrauliformis), common Kilka (C. cultriventris caspia), and bigeye Kilka (C. grimmi) (Fazli et al. 2009). Common Kilka has been identified as one of the most important industrial and commercial fish in the Caspian Sea. A total catch of 24, 000 tons of common Kilka was reported by the Iranian Fisheries Organization for the year 2010 (Shilat 2010). Unfortunately, a small portion of the Kilka catch is used by human (4%) due to its small size, easy deterioration, impossibility of gutting right away after catching, difficulties in hygienic preservation, packaging, and supply and the rest is used as fish meal for poultry and in aquaculture (Khoshkhoo et al. 2010;Khanipour et al. 2014). Consequently, to increase the amount of consumption, it is proposed to have various fish products derived from it. Today, production of minced fish which are vastly consumed could be one way of increasing fish consumption (Asgharzadeh et al. 2006). It is an intermediately ready product for preparation of different highly acceptable ready to serve seafood products like frozen surimi, frozen mince block, extruded product, imitated products, etc. (Devi et al. 2013).
Spoilage of fish results from changes brought about by biological reactions such as oxidation of lipids, the activities of the fish muscular enzymes, and the metabolic activities

ORIGINAL RESEARCH
Comparing the effect of encapsulated and unencapsulated fennel extracts on the shelf life of minced common kilka (Clupeonella cultriventris caspia) and Pseudomonas aeruginosa inoculated in the mince of microorganisms. Minced fish is more susceptible for microbial spoilage than whole fish due to greater surface area of minced fish. Food-borne infections are one of the most serious and costly public health concerns worldwide. Increasing demand for high-quality fish products with extended shelf life has initiated the development of several innovative techniques to keep quality attributes as long as possible and yield safe products (Maftoonazad and Badii 2009).
Lightly preserved food products with natural additives have become popular due to the modern trends adopted by consumers toward the consumption of food and seafood free from pathogens, with minimally processed foodstuffs containing no chemical preservatives (Abdollahzadeh et al. 2014). Fennel (Foeniculum vulgare) is one of the most important medicinal and aromatic plants due to its estrogenic activities and applications as a carminative, diuretic, antiinflammatory, antimicrobial, and galactogogue; it is a substance which is used to increase the production of milk in humans and animals (Mahfouz 2007). Fennel is also used for its medicinal properties; it is nowadays cultivated for industrial uses such as cosmetic and pharmaceutical products (El Ouariachi et al. 2014). Foeniculum vulgare extract demonstrated bacteriostatic activity against the following bacteria using the agar diffusion and serial dilution method: Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa (Bone and Mills 2013). It has shown antimicrobial (Gulfraz et al. 2008) antioxidant activity in various in vivo and in vitro experiments (Barros et al. 2009;El Ouariachi et al. 2014), its ability in fish preservation has not been studied.
However, plant extracts generally present some formulation problems, such as long-term instability, low bioavailability, and a significant burst release. Therefore, there is a need for their protection. Encapsulation is proposed as a convenient method for that purpose (Rijo et al. 2014). It was already explained that, encapsulation can protect antioxidants from light and oxygen, and preserve their stability and activity to some extent (Belščak-Cvitanovic´ et al. 2011;Bojanaa et al. 2013). Some studies (Gortzi et al. 2007;Bojanaa et al. 2013) have also shown that encapsulation can improve antimicrobial and antioxidant activity of compounds and maintain the stability of them over prolonged periods of time.
Thus, this study was aimed to investigate the effect of encapsulated and unencapsulated fennel extract on the quality of minced common Kilka during storage at refrigeration condition (4 ± 1°C) and behavior of Pseudomonas aeruginosa inoculated to it.

Materials
The plant fennel (Foeniculum vulgare) was purchased from a local market and authenticated by the Medical Plant Incubator Center (Islamic Azad University, Ayatollah Amoli Branch, Amol, Iran). The solvent (ethanol) was added to powdered fennel in ratio of 1:10 and the resulting mixtures were shaken overnight to extract fennel's phenol compounds. After 24 h, the extracts were filtered through Whatman No. 42 filter paper to separate thyme particles. The solvents were completely evaporated in an oven at 40°C. Finally, they were placed in a refrigerator (Esmaeilzadeh Kenari et al. 2014). Pseudomonas aeruginosa was purchased from the Iranian Research Organization for Science and Technology (Persian Type Culture Collection (PTCC), Tehran, Iran). Gum arabic was obtained from Sigma-Aldrich Chemical Co (St Louis, MO, U.S.). All other chemicals were of analytical grade.

Encapsulation by spray drying
Encapsulation of the fennel extract with gum arabic was done according to the method described by Beristain (2001) with some modifications. Solutions of gum arabic were prepared by dissolving 300 g (w/w) of gum arabic powder in distilled water. The obtained solutions were heated at 60°C with constant stirring for 20 min, covered, and left overnight at room temperature. Fennel extract was added to the hydrated gum solution at extract: gum ratio of 1:4 w/w with respect to gum solids. The mixture was homogenized with an Ultra-Turrax T50 homogenizer (IKA-WERKE Works Inc., Wilmington, NC) at 7000 rpm for 15 min and fed into a Buchii Mini Spray Dryer model 190 (Buchii Laboratoriums-Technik AG., Flawil, Switzerland). The drying condition inlet air temperature was 105°C and the outlet air temperature was 108°C with a feeding rate of 5.6 mL/min. Encapsulated and unencapsulated fennel extract was used in the preparation of 0.3% and 0.5% (w/v) solution in distilled water of 4°C.

Fish sample preparation and storage condition
Fifty live commercial-sized Kilka with an average weight 7 g were purchased from a public market alive and transferred to the Caspian Sea Ecology Research Center in Iran in sealed and foamed polystyrene boxes containing flaked ice. Then the fishes were killed by slurry ice, washed by hand, and minced twice. Furthermore, a ranking test previously carried out comparing fish samples with encapsulated fennel at different concentrations showed significantly lower acceptability of the samples incorporating 0.75 or 1% encapsulated fennel when compared to the rest (0.5% or lower) (data not shown). After these results, the encapsulated fennel concentration of 0.3% and 0.5% was chosen as optimal for the following study of fish preservation, and then minced fish was randomly assigned into five batches (100 ± 10 g minced fish in each group). The first batch was prepared without fennel extract (control batch), four of the batches processed with unencapsulated and encapsulated fennel extract as following; batch 2 (0.3% unencapsulated RE), batch 3 (0.5% unencapsulated RE), batch 4 (0.3% encapsulated RE), batch 5 (0.5% encapsulated RE). For Pseudomonas aeruginosa analysis, minced samples were inoculated with 1 × 10 4 CFU/g of Pseudomonas aeruginosa. Then, the encapsulated and unencapsulated fennel extract were added according to the above-mentioned treatments. After packaging, all samples in the polyethylene dishes were covered with a cellophane blanket, they were stored at 4 ± 1 °C for subsequent quality assessment. Chemical and microbiological analyses were performed at 3 day intervals to determine the overall quality of the minces.

Chemical analysis
The peroxide value (PV) of the samples was determined in the total lipid extracts according to the method of Pearson (Egan et al. 1997). Results were expressed in meq oxygen kg-1 lipids.
The total volatile basic nitrogen (TVB-N) of the samples was determined by the microdiffusion method as described by Goulas and Kontominas (2005). Results were expressed in mg N/100 g of fish and performed in triplicate.

Microbiological analysis
Bacteriological counts were determined by placing a 10 g of the mince sample in 90 mL of physiological serum, and homogenizing with a stomacher. The total viable count (TVC) and total psychrotrophic count (TPC) of the samples were determined by the pour plate method, using plate count agar (PCA, Merk, Darmstadt, Germany). The inoculated plates were incubated at 37°C for 2 days for TVCs, and at 10 °C for 7 days for psychrotrophilic counts)AOAC 2005(. For Pseudomonas aeruginosa enumerations, 0.1 mL from 1:10 prepared serial dilutions (0.1% physiological solution) of fish mince homogenates was spread onto the surface of solid media. Pseudomonas was determined on Pseudomonas Isolation agar (PIA, Oxoid, UK) after incubation at 48 h at 35 °C. This medium is selective and formulated to enhance the formation of blue or blue-green pyocyanin pigment by P. aeruginosa (Pavelková et al. 2014). All counts were expressed as log colony-forming units (CFU/g) and performed in triplicate (Ibrahim Sallam 2007).

Statistical analysis
One-way ANOVA was used and mean comparison was performed by Duncan's new multiple range test. Statistical analysis was prepared using the SPSS statistical software, (release 18.0) for Windows (SPSS Inc. Chicago, IL). All data are presented as mean ±SD. Significant differences were considered at the 95% confidence level (P < 0.05).

Results and discussion
Changes in peroxide value (PV) content The effects of encapsulated and pure fennel extract on the changes in the PV content of the minced Kilka during 15 days storage at 4°C are shown in Figure 1. The peroxide value which detects a measure of the concentration of peroxides and hydroperoxides formed in the initial stages of lipid oxidation is widely used for the estimation of oxidative rancidity in fats (Ólafsdóttir et al. 1997). The PV values of samples significantly increased (P < 0.05) during storage time, until day 9, and then decreased gradually until the end of storage time. After the maximum value was reached, a decrease in PV values was observed. This decrease is an unequivocal sign of advanced oxidation (Pereira de Abreu et al. 2010; Ariaii et al. 2014). A slower increase in PV values was obtained in samples treated with encapsulated and unencapsulated FE, in contrast to a faster increase in PV of the control sample after 3 days of storage time, demonstrating the oxidative stability of fish lipids by fennel extract. These results could be attributed to the antioxidant activity of fennel extract which is related to its polyphenol contents. This observation was in agreement with what reported by Chang et al. (2013) about the antioxidant properties of fennel extract. They showed that fennel extract had antioxidant effect on lipid stabilization by delaying the hydroperoxides formation. At the end of storage time, the maximum and minimum PV were related to control (6.1 meq oxygen kg-1 lipids) and 0.5% encapsulated FE (3.76 meq oxygen kg-1 lipids), respectively. This indicated the potential of encapsulation to improve the antioxidant activity of the fennel extract during application in minced Kilka by prolonging its availability. Evidence of encapsulation improving the bioactivity and bioavailability of polyphenols has been reported by a number of researchers (Fang and Bhandari 2010). Furthermore, the results indicated that a high concentration of encapsulated FE was more effective in controlling oxidation. However, it has been reported that a higher concentration of extract may act as a better pro-oxidant (Sarah et al. 2010).

Changes in the total volatile basic nitrogen (TVB-N)
The TVB-N value is one of the most widely used indicators of seafood deterioration. It is a general term which includes the calculation of three methyl amine (produced by spoilage bacteria), dimethyl amine (produced by autolytic enzymes during chilled preservation), ammonia (produced by the deamination of amino acids and nucleotide catabolizes) as well as other volatile basic nitrogenous compounds correlated with seafood spoilage (Erikson et al. 2011). The effects of encapsulated and unencapsulated fennel extract on the changes in the TVB-N content of the minced Kilka during 15 days storage at 4°C are shown in Figure 2. The initial TVB-N value of the fillets was 10.66 mg/100 g which showed the good quality of fresh samples in that, freshwater fish muscle has 10-20 mg/100 g TVB-N after harvesting (Alçiçek 2011). The value of TVB-N increased progressively with the time of storage for all fish samples. TVB-N increase in the control group was higher than fennel extract added groups during storage. After 3 days, the difference between the control group and the others was found to be significant (P < 0.05). At the end of storage time, the maximum and minimum TVB-N were related to control (59.78 mg/100 g) and 0.5% encapsulated FE (38.76 mg/100 g), respectively. Since TVB-N is produced mainly by bacterial decomposition and endogenous enzymes of fish muscle, the lower value of TVB-N observed in samples treated with fennel extract can be related to the antibacterial activity of the extract which would more rapidly reduce the bacterial population and this could be due to the decreased capacity of bacteria for oxidative deamination of nonprotein nitrogen compounds (Banks et al. 1980). According to Connell (1990), a level of 35-40 mg TVB-N/100 g of fish flesh is usually regarded as spoiled. The TVB-N values of the samples in our study exceeded the maximum level by day 6 for control, by day 9 for 0.3% FE, and by day 12 for 0.5% FE and 0.3% encapsulated FE. It is lower than the acceptable limit for 0.5% encapsulated FE till the end of storage time. Anwar et al. (2009) reported appreciable antimicrobial activity for fennel extract and essential oils against selected strains of bacteria and pathogenic fungi. On the other hand, samples treated with gum arabic encapsulated fennel extract showed significantly lower TVB-N content compared to control and fillets treated with pure extract during the storage period (P < 0.05). This observation may be explained by the enhanced antimicrobial activity of the extract after encapsulation or the better protection of their functionality during the processing or storage period (Gortzi et al. 2007).

Microbiological changes
The composition of fish muscle makes it favorable for microbial growth. Thus, fish spoiling occurs during storage mainly as a result of microbial activity (Souza et al. 2010). Changes in the total viable count (TVC) and total psychrotrophic count (TPC) of minced Kilka are shown in Figure 3A and B. The initial TVC and TPC of fish sample was 3.5 and 3.1 log CFU/g, respectively, and the low initial TVC and TPC indicated very good fish quality (Fan et al. 2009).
Total viable count of all samples increased with storage time and the value of control increased faster and exceeded the maximum of 10 7 log CFU/g after 6 day. This acceptability limit of 10 7 CFU/g has been recommended for fresh fish (ICMSF, 1986). As can be seen, all minced samples treated with encapsulated and unencapsulated FE significantly inhibited the growth of mesophilic bacteria compared with the control during the storage period. These results showed antimicrobial properties of FE as a well known source of main component, such as contained (E)-anethole, limonene, apiole, beta-fenchyl acetate, and perillene known to exert antimicrobial activity (Cetin et al. 2010). The total viable count of 0.5% encapsulated FE samples was lower than 7 CFU/g till the end of storage time. Significant changes were observed in microbial analysis of pure fennel samples and encapsulated fennel extract samples (P < 0.05). A similar trend was also observed about TPC in all treatments. This indicated that encapsulated fennel extract could strongly inhibit the growth of TVC. The improvement of the antimicrobial activity of natural plant extracts and essential oils when encapsulated was also reported by others (Gortzi et al. 2006(Gortzi et al. , 2007Liolios et al. 2009;Donsì et al. 2011).

Inhibition the growth of Pseudomonas aeruginosa in minced fish
The effects of encapsulated and unencapsulated fennel extract on Pseudomonas aeruginosa inoculated in Kilka mince are presented in Figure 4. As can be seen, the initial population of Pseudomonas aeruginosa in the control group increased rapidly during the storage period. Although, the sample treated with encapsulated and unencapsulated fennel extract showed that a population of Pseudomonas aeruginosa significantly (P < 0.05) lowers than the control. Although the sample treated with unencapsulated FE at 0.3 showed that a population of Pseudomonas aeruginosa significantly lowers than the control, but it could not inhibit its growth completely during the storage period. Unencapsulated FE at 0.5% and encapsulated FE at 0.3% could significantly reduce the population of Pseudomonas aeruginosa compared to the control-and 0.3% FE-treated samples. These results were in agreement with those reported by Careaga et al. (2003) about the effect of Capsicum extract on Pseudomonas aeruginosa inoculated in raw beef meet. They reported that raw beef samples treated with Capsicum extract at 0.3% exhibited the lower population of Pseudomonas aeruginosa, as compared with the untreated. Nevertheless, the drastic bactericidal effect of Capsicum extract was observed, when 4 or 5% extract was used, which was approximately 10-fold more than the amount used in this study.
From day 6 until the end of storage, the degree of decline in the Pseudomonas aeruginosa population in 0.3% encapsulated FE samples was significantly (P < 0.05) greater than in the unencapsulated FE 0.5% minced fish samples. According to the study, drastic bactericidal effect of fennel extract was observed in the encapsulated FE 0.5% minced fish samples during the mentioned days . These coincide with our observation about the TVC and TPC of minced Kilka which reveals the efficacy of encapsulation in improving the antimicrobial properties of FE.


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Thyroid Hormones in Donkey Blood and Milk: Correlations with Milk Yield and Environmental Temperatures

Thyroid hormones (TH) are the primary endocrine stimulators of non-shivering thermogenesis and are known to stimulate lactation. Triiodothyronine (T3) is the bioactive form, mainly derived by deiodination of thyroxine (T4), and the free quote (unbound to plasma proteins) is immediately bioavailable. This study aimed to evaluate potential relationships among TH in the blood, triiodothyronine in the milk (T3M), milk yield and environmental temperature in March to July for 8 lactating donkeys. Milk yield and blood TH concentrations changed significantly over time, whereas T3M was rather stable among individuals and not affected by time of sampling. Free T3 was not correlated with free T4 or with total TH in the blood, but it was weakly correlated with T3M. No relationship was found between blood TH and milk yield, which was negatively correlated with T3M. Thus, the absolute quantity of bioactive hormone in milk secretion is maintained. Milk yield was positively correlated with the free/total T3 and free T3/free T4 ratios, thus in turn with the relative quote of the circulating bioactive hormone. Circulating T3/T4 ratios were negatively correlated with environmental temperature. It is concluded that environmental temperature, in the range of the present study (-2 to 35°C), does not significantly entrain thyroid gland activity, which is affected more by other factors, such as inter-individual variations and physiological status (i.e., stage of lactation). However, increases in environmental temperature most likely induce decreases in deiodinase activity at the peripheral tissue level, as indicated by the decrease in the T3/T4 ratios in the blood.

Introduction
The thyroid hormones (TH), tetraiodothyronine or thyroxine (T4) and 3-3'-5-triiodothyronine (T3), act stimulating oxygen utilization and heat production in every cell of the body. The overall effects of these hormones are to increase the basal metabolic rate, make more glucose available to cells, stimulate protein synthesis, increase lipid metabolism and stimulate cardiac and neural functions. The thyroid gland, as such, secretes mostly T4, which is monodeiodinated to T3, the active form that binds to receptors at the target cells and exerts its biological effect (Capen and Martin, 1989). More than 99% of TH circulates in the blood bound to plasma proteins. Only the free hormone is responsible for the biological activity, and protein-bound hormones function as a promptly utilizable source, delaying the effects of decreased thyroid secretion and buffering against sudden increases in the thyroid's secretory activity (Bartalena, 1990;Utiger, 1995). Thyroid hormones are the primary endocrine stimulators of non-shivering (facultative or adaptive) thermogenesis, thus regulating body temperature (Silva, 2005). A major exogenous regulator of thyroid gland activity is environmental temperature (Dickson, 1993); therefore, an inverse relationship between ambient temperature and blood TH concentrations often has been found in many animal species, such as domestic ruminants (Todini, 2007). Thyroid hormones are known to stimulate lactation (Tucker, 2000) and play a permissive role for galactopoietic hormones, such as prolactin and somatotropin (Kahl et al., 1995). During lactation, TH (together with insulin and cortisol) drives nutrient partitioning toward milk production and visceral protein synthesis (Motil et al., 1994). On the other hand, active T3 in colostrum and milk could play various physiological roles, such as serving as a source of systemic T3 and as a paracrine action that supports lactogenesis in the mother; on the other hand, physiological effects on the suckling offspring may be systemic and/or occur within the gastrointestinal tract (Sheard and Walker, 1988;Murray and Luba, 1993;Slebodzinski et al., 1995).
The ancient traditional utilization of donkeys (Equus asinus) as dairy animals excited a renewed popular and substantial scientific interest with regard to human nutrition in recent years. Clinical evidence shows that donkey milk is well tolerated by infants who are allergic to cows' milk (Monti et al., 2007;Vita et al., 2007), and its use is reported to be use-ful in the treatment of human immune-related diseases and in the prevention of atherosclerosis (Tafaro et al., 2007). Donkey milk gross composition is similar to human milk, except for the low and variable average total solids and fat content (Salimei, 2011). Donkey mammary secretions contain compounds that can exert biological activities (i.e,. IGF-1, leptin, ghrelin and T3) . Moreover, Todini et al. (2012) reported that in donkey milk, the concentration of T3, a human-like bioactive compound, can be affected by the supply of dietary trace elements.
As part of the search for potential factors that entrain thyroid gland activity and TH bioavailability in donkeys, the present study aimed to identify relationships among TH concentrations in lactating donkeys, milk production and environmental temperatures.

Location, animals and diet
The animal care procedures followed the European recommendations for the protection of experimental animals (EU Directive 2010/63/EU; European Commission, 2010).
The study was carried out on a farm in the Emilia Romagna region, Italy, at an altitude of 317 m a.s.l. and at latitude 44.38 °N, where approximately 500 donkeys are bred for dairy purposes.
Eight lactating, pluriparous jennies (6-11 years old), at 34-57 days post partum at the beginning of the study, were used to provide blood and milk samples. The day of samplings (every two weeks) they were weighted and their body condition scores were measured (on a 1 to 5 scale: Martin Rosset, 1990). Animals had continuous access to fresh water and to meadow hay and were fed 2.5 kg of mixed feed (12.6% crude protein as fed basis), divided into two meals. Jennies were housed with foals, which were separated from the dams 3 h before mechanical milking (Salimei et al., 2004).
Environmental temperatures were measured by a meteorological station near the farm and recorded by the Parma station. The following data were considered: maximum, minimum and mean temperatures of the sampling day, and the respective means for the 7 days preceding the samplings.

Samplings and assays
Individual milk and blood samples were collected every 2 weeks from 9 March to 1 June 2009 and then on 9 July 2009 (eight sampling times) at 1100 a.m. during the mechanical milking session (Salimei, 2011). Individual milk yield was recorded by graduated bulk, and the values were also expressed for yield relative to body weight (mL/kg body weight/milking). Because foals were approaching weaning, milk production at the last sampling date was scanty and was not considered for elaboration. Aliquots of milk samples were utilized for iodothyronine extraction with alkaline ethanol at low temperature, as described by Slebodzinski et al. (1998) and by Todini et al. (2010). Briefly, 2 mL of cold (-20°C) alkaline ethanol (pH 9.0 with NH4OH) were added to 1 ml of whole donkey milk, mixed thoroughly with a glass rod, vortexed and left in the freezer (-20°C) for 24 h. Then, the milk-ethanol mixture was vortexed, left in the freezer for a further 24 h and then centrifuged at 3500 g at 0°C for 30 min. The supernatant was collected and stored at -20°C. Twenty-four hours before the assay, these supernatant samples were diluted 1:1 with the assay buffer.
Blood samples were collected by jugular venipuncture in evacuated tubes containing K3-EDTA as anticoagulant (Venoject; Terumo Europe NV, Leuven, Belgium). Tubes were immediately centrifuged (2500 g for 15 min), and the plasma aliquots were stored at -20°C until assayed. The total concentrations of T3 in the extracted milk samples (T3M), total T3 and T4 in blood plasma (TT3P and TT4P), and free (unbound) T3 and T4 in blood plasma (FT3P and FT4P) were assayed using commercially available enzyme immunoassay (EIA) kits that are competitive ELISAs designed for clinical use in humans (T3KT1EW, T4KT2EW, FT3KT0037EW, FT4KT7EW; Radim, Rome, Italy). Assays were performed using the automated processor Brio 2 reader (Seac, Firenze, Italy). The assay kits were specifically validated for donkey species, as previously described (Todini et al., 2010). Intra-and inter-assay coefficients of variations were all between 1.9 and 8.4%. For each hormone, all samples from one animal were analyzed within the same assay session. Hormone concentrations were determined as the mean of duplicate determinations. The ratios of free/total hormones were also calculated, as well as the ratios of T3/T4 (both free and total). As a part of a larger study on lactating donkeys, data about changes of total TH with time have been partially published elsewhere (Todini et al., 2012).
During the trial, fresh bulk milk samples were analyzed by IR for crude protein, fat, and lactose content, and gravimetrically for total solids and ash content (Milkoscan, Foss, Hillerod, Denmark). The health of the mammary glands and milk hygiene were also monitored by somatic cell count (Fossomatic 360, Foss, Hillerod, Denmark) and total bacteria count (Bactoscan 8000, Foss, Hillerod, Denmark) in fresh bulk milk samples.

Statistical analysis
Statistical analyses were performed with SPSS 12.0 (SPSS Inc., Chicago, IL, USA; SPSS, 2003). All the investigated variables were checked for normal distribution using the Kolmogorov-Smirnov test. To meet ANOVA assumptions (i.e., normal distribution and homogeneity of variance) data were logarithmically (FT3P, TT4P, TT3P, T3M) or square-root (FT3P/TT3P, FT3P/FT4P, FT4P/TT4P, TT3P/TT4P) transformed for statistical elaboration. The results presented in tables and figures are least square means ± SE from the original data. The data were processed by analysis of variance for repeated measures to evaluate the effects of time of sampling.
Differences between means were analyzed by least significant difference. Associations among variables were examined by calculating simple linear correlation (Pearson coefficient). Significant correlations were declared as strong (r>0.7), moderate (r from 0.3 to 0.7) or weak (r<0.3).

Results
At the beginning of the experiment the jennies have mean body weights of 205±32 kg and 3.45±0.39 body condition score, both of which did not significantly change during the trial.
Data from bulk milk samples indicated that the health of the udders (somatic cell count 8200/mL), milk hygiene (colony forming unit 3600/mL) and milk gross composition (crude protein 1.74%, fat 0.24%, lactose 7.05%, ash 0.40%) were within the ranges previously reported for donkey milk . In agreement with previous studies, the milk protein content significantly (P<0.05) decreased during the trial (from 1.87 to 1.59%) but the fat and lactose content remained constant .
Means for milk yield and TH concentrations are reported in Table 1 and the respective trends during the study are described in Figure  1. There was a significant (P<0.05) effect of time on milk yield and blood TH concentrations, whereas T3M was not affected. The free/total hormone concentrations ratios were affected by time (P<0.01 and P<0.05, for T3 and T4, respectively). The rates of bound circulating hormones ranged from 99.9991 to 99.9999% for T3 and from 99.9897 to 99.9997% for T4.
Correlations among hormone concentrations and milk yield are reported in Table 2. Total T3 and T4 in the blood were strongly correlated (r=0.8, P<0.0001). Free T4 in blood was strongly correlated with TT4P (r=0.72, P<0.0001) and TT3P (r=0.72, P<0.0001). Free T3 in blood was not correlated with TT3P, TT4P or FT4P, but it was weakly correlated with T3M. None of the hormone concentrations in the blood (TT3P, TT4P, FT3P, FT4P) were correlated with milk yield/milking (actual or expressed for body weight), but T3M was negatively correlated with the actual milk yield per milking. Milk yield/kg body weight was positively correlated with the FT3P/TT3P and FT3P/FT4P ratios. Figure 2 shows the mean daily environmental temperature and the ratios of T3/T4 in the blood. There was a significant effect of time on circulating T3/T4 ratios (P<0.05 and P<0.01 for TT3P/TT4P and FT3P/FT4P, respectively).
The correlations between thyroid hormone concentrations and milk yield with environmental temperatures are reported in Table 3. Milk T3 concentration showed moderate positive correlations with all the temperature parameters. Free T3 in blood was negatively correlated with the mean and and maximum temperatures of the day of sampling. The ratios TT3P/TT4P and FT3P/FT4P were negatively correlated with almost all the environ-mental temperatures, moderately but sometimes very significantly. Milk yield was negatively correlated with some temperature parameters.
t i a l binding affinities of the plasma proteins, in particular for the active hormone T3, which also showed a very wide range in the free/total hormone ratio (Table 1). The plasma concentrations of free T3 were not correlated with the concentrations of total TH and free T4 (Table  2). This could indicate that the effective amount of quickly bioavailable hormone is finally regulated and dependent on other factors mainly at tissue level, beyond the thyroid gland activity. Both T3 and T4 free/total ratios (Figure 1d) showed profiles similar to those of the total hormone concentrations (Figure 1b  and 1c), with the respective peaks often coinciding; this may indicate that a certain degree of saturation of the plasma binding protein can occur. The percentages of bound fractions, which were well above 99.98% for both hormones, were similar to those found in pregnant and barren donkeys, by the same ELISA methods (Fazio et al., 2012). It is noteworthy that the concentration of active T3 in milk was less variable among indi-  viduals and did not significantly change with time (Table 1). In mare milk lower levels than in our donkeys have been obtained by RIA: 0.5-0.76 ng/mL, associated with a mean of 0.9 ng/mL in maternal serum, giving an approximate colostrum/serum ratio of 0.82 (Slebodzinski et al., 1998). At a mean colostral T3 concentration of 0.456 mg/L and a daily intake of colostrum by a foal of about 4 L, the daily T3 consumed by a suckling foal is approximately 1.8 mg, which may likely have some stimulatory effects within the intestinal tract (Slebodzinski et al., 1998). Triiodothyronine concentration in milk is considered mainly dependent on deiodinase activity at the mammary level, as in mares they are strongly correlated (Slebodzinski et al., 1998). The correlation between free T3 in the blood and T3 in the milk (Table 2) may indicate a certain transfer of active T3 from blood to milk, although no conclusions can be advanced from the current study.

Iodothyronines in donkey blood and milk
During the current experiment, thermal conditions were not extreme and did not fall in the values commonly considered for heat stress (Silanikove, 2000). On many occasions, the maximum daily temperature was above 30°C, but the minimum during the night was always under 20°C ( Figure 2a). Nonetheless, milk yield was negatively correlated with some parameters of environmental temperature (Table 3), which rose along with advancing season and stage of lactation ( Figure 2a). Therefore, under the conditions of the present experiment, the potential effect of relatively high temperature on the milk yield decrease, as observed with advancing lactation, remains to be demonstrated. Milk yield showed no correlation with hormone concentrations in the blood, but was positively correlated with the FT3P/TT3P and FT3P/FT4P ratios (Table 2), thus with the relative quote of the bioactive hormone, according the well known stimulatory role of general metabolism and lactogenesis in particular (Tucker, 2000). The negative correlation between milk T3 concentrations and milk yield per milking (Table 2) could either be due to a simple dilution effect or suggest a homeostatic control for maintaining an absolute quantity of active hormone in milk secretion. Therefore, concentrations of T3 in milk tended to increase when milk yield was decreasing, and this corresponded with advancing lactation and the spring season, and thus, to increasing environmental temperatures. We can only hypothesize such a mechanism as an explanation for the positive correlations found between milk T3 and temperature parameters (Table 3).
In the mild conditions of the present study, environmental temperature had weak or no effect on blood hormone concentrations, which mainly reflect gland secretion. Serum T4 showed no correlation with ambient temperature down to -31°C in Icelandic horses (Mejdell and Boe, 2005), and Cymbaluk (1994) had previously also reported that horses do not respond to chronic cold exposure by increasing thyroid hormone secretion, which instead is stimulated more by lengthening photoperiod.
In the present study, the supposed inhibitory effect of the increasing environmental temperature may have been overcome by the increasing day length. In fact, treatment with artificial photoperiod cycles clearly affected concentrations of circulating TH (Todini et al., 2006) and the light-induced increase in circulating hormones could play a role in sustaining advanced lactation. The galactopoietic effect of increasing day length have been demonstrated in dairy ruminants submitted to artificial photoperiod (Dahl et al., 2000(Dahl et al., , 2015. In goats, short-day treatment during the last third of pregnancy induced higher blood T3 afterwards during the next lactation, accompanied by higher milk yield (Mabjeesh et al., 2007). A retrospective study, based on a very large number of cow milk samples collected during several years, allowed to establish critical limits of temperature humidity index values for mammary health and milk composition (Bertocchi et al., 2014). The significant inverse relationship between environmental temperature and T3/T4 ratios (both free and total) (Table 3) indicates that regulation of peripheral deiodination allows enough prompt and fine adjustments of bioavailability and activity at the target level, without significant changes in thyroid gland secretory activity. In ruminants, even in conditions of heat stress, the response of the thyroid gland is rather slow and the effects, such as the decline of blood TH concentrations, are only visible after several days (Silanikove, 2000). Also, the stimulatory effect of day length on blood T4 in goats was characterized by a long latency of several weeks (Todini et al., 2006). In this view, the positive correlations between T4 and minimal environmental temperatures (Table 3) can be explained; the adjustments of thyroid gland secretory activity, for example in case of cold exposure, are likely preceded by a short-term stimulation of peripheral deiodination, which consumes circulating thyroxine, as reflected by the proportional increases of T3 and T3/T4 ratios. Vice-versa, in animals facing a hot climate, an inhibition of deiodinase activity occurs and less T4 is activated to T3. Therefore, whereas FT3 was negatively correlated with the daily mean and maximum tem-perature, such an inverse relationship was much more evident and constant for the T3/T4 ratios, for both free and total hormone levels (Table 3).

Conclusions
In conclusion, the concentrations of the bioactive triiodothyronine in donkey milk are not negligible and do not vary between individuals and over time, unlike the hormone concentrations in the blood. Moreover, the inverse relationship between milk T3 concentration and milk yield may indicate that adequate amounts of the hormone are maintained and can be acquired by the suckling foal, even when milk yield decreases, i.e., with advancing lactation and toward weaning.
We also conclude that environmental temperature, in the range of the present study (-2 to 35°C), does not significantly affect thyroid gland activity. However, an increase in environmental temperature most likely induces a decrease in the deiodinase activity at the peripheral tissue level, as indicated by the decrease in the T3/T4 ratios in the blood.
The hormone bioavailability and effects at the target level can be regulated by changes in thyroid gland secretion, activity of deiodinase enzymes, and plasma binding proteins. At each of these levels, many endogenous, as well as exogenous, regulatory factors can act. Considering that donkeys in the world are most commonly located in areas characterized by a harsh climate, thyroid hormone physiology is a main topic among the mechanisms that allow the animals to adapt to the surrounding environment, thereby maintaining their welfare and productions.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

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Antimicrobial and Antioxidant Activities and Phenolic Profile of Eucalyptus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill & L.A.S. Johnson Leaves

This study was performed to evaluate the in vitro antimicrobial and antioxidant activities and the phenolic profile of Eucalytus globulus Labill. and Corymbia ficifolia (F. Muell.) K.D. Hill & L.A.S. Johnson leaves. Both leave extracts contain significant amounts of phenolic compounds, mainly flavonoids. Qualitative and quantitative analyses of the phenolic compounds were performed using a HPLC/MS method. The main flavonoid was hyperoside and its highest amount was found in E. globulus (666.42 ± 5.02 μg/g dw plant material). Regarding the flavonol profile, myricetin was the dominant compound and its highest amount was found in C. ficifolia leaves (124.46 ± 0.24 μg/g dw plant material). The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays, revealing an important antioxidant potential for both species. In the antimicrobial assays, C. ficifolia extract was found to be more active than E. globulus against both Gram-positive and Gram-negative bacterial strains with the exception of Bacillus subtilis. The results of the present study provide new valuable data regarding the bioactivities of these medicinal species.

Introduction
Historically, the human pharmaceutical arsenal is significantly indebted to Nature and in particular to natural products obtained of traditional medicinal plants, fungi and bacteria [1]. To date, natural products and compounds derived therefrom command a substantial market share, comprising 61% of anticancer compounds and 49% of anti-infectives approved in the past 30 years [2]. In medicinal plants, these compounds are considered as bioactive natural products and may ultimately be developed as drugs. In food, they would be defined as phytonutrients without therapeutic claims but with significant health benefits that can be used in disease prevention [3].
Under the name of "Gum trees" or "Eucalypts" there are included, from a taxonomic point of view, three genera, namely Eucalyptus L'Heritier (1789), Angophora Cavanilles (1797) and Corymbia Hill & Johnson (1995) [4,5]. The genus Eucalyptus s.str. encompasses more than 500 species native mostly to Australia with some species also native in New Guinea and the Philippines [5]. One of the best known species is Eucalyptus globulus Labill. or "Blue Gum" which is native to south-eastern Australia and Tasmania [5], but also widely cultivated in the Mediterranean region [6]. Its leaves have been used as traditional remedies for treatment of various diseases such as pulmonary tuberculosis, influenza, fungal infections and diabetes [7][8][9][10]. Because of their antioxidant activity, leaf extracts of E. globulus have been also used as food additives [9].
The genus Corymbia consists of 113 species which are generally named "bloodwoods" due to their propensity to exude a red gum from the cut bark. All of them are endemic to Australia and can be distinguished from the related genera by their corymb inflorescences [11]. The most well-known and the most cultivated species of the genus is Corymbia ficifolia (F. Muell.) Hill et Johnson (previously known as Eucalyptus ficifolia) despite the fact that it is a local endemic of the Walpole region of southern part of Western Australia [12]. Even though Corymbia species have been mostly used as ornamental trees, recent research has suggested medicinal usage possibilities [13].
Most of the research conducted on Eucalyptus has mainly focused on the composition and biological activities of the essential oil extracted from the leaves, revealing important antibacterial properties [6,7,10,14]. However there are some isolated studies that mainly deal with the chemical composition of the leaf extracts and their antimicrobial and antioxidant properties [7,15]. Literature data on Corymbia species consists of just a few reports that describe the chemical composition of the leaf essential oil from Corymbia citriodora Hook. or other species [16,17] or the composition of fruit resins and their antibacterial properties [13,18], but no data was found regarding C. ficifolia.
Nevertheless, recent studies on Eucalyptus and Corymbia report a positive correlation between the antioxidant and antimicrobial properties and the polyphenolic content of stumps and fruits [8,9,13,19]. The importance of phenolic compounds has increased due to the linkage between oxidative stress and the development of chronic diseases, including coronary heart disease, cancer as well as aging. Recently, phenolics have been considered powerful in vitro antioxidants and proved to be more potent antioxidants than vitamins C and E and the carotenoids [20]. In this context, further comprehensive studies on polyphenolic compounds and their amounts in plants are essential. A rapid and sensitive HPLC-UV method assisted by mass spectrometry detection was utilized for the determination and quantification of polyphenols in the leaves extracts of these species. The overall aim of this paper was to conduct a comparative study of the chemical composition of E. globulus and C. ficifolia leaves and to evaluate their antioxidant and antimicrobial activities, for a better usage and characterization of these medicinal herbs.

HPLC-UV-MS Analysis of Polyphenols
The employed method was developed for the identification and quantification of nineteen phenolic compounds: eight phenolic acids and eleven flavonoids. The quantitative determination was performed by the external standard method and the concentrations of the identified phenolic compounds are shown in Table 1 in order of their retention times. Gentisic, chlorogenic and p-coumaric acids were identified in the C. ficifolia extract, but none of these compounds could be quantified. The only phenolic acid present in the E. globulus extract was chlorogenic acid.
The patterns of flavonoids and flavonols indicate large quantitative differences. The dominant flavonoid in both extracts is hyperoside, with its highest amount being found in E. globulus (666.42 ± 5.02 μg/g dw plant material). Isoquercitrin, rutin and quercitrin were found in both extracts and the highest amount was seen in C. ficifolia (71.31 ± 0.07, 180.77 ± 0.21 and 334.57 ± 0.35 μg/g dw plant material, respectively).
Regarding the flavonol profile, myricetin is the main compound in both extracts, and it is found in a higher quantity in C. ficifolia (124.46 ± 0.24 μg/g dw plant material). The flavonol with the lowest amount in both extracts is quercetin (Table 1), the richest species also being C. ficifolia (5.04 ± 0.04 μg/g dw plant material). Pereira et al. reported higher quantities of quercetin and luteolin (206.51 ± 31.11 and 161.57 ± 24.25 μg/g dw plant material, respectively) in a 70% methanol-aqueous extract [7].
The polyphenols from E. globulus and C. ficifolia leaves were analyzed. In summary, the simultaneous determination of a wide range of polyphenolic compounds was performed using a rapid, highly accurate and sensitive HPLC method assisted by mass spectrometry detection, and the comparative study showed large quantitative differences between the two species.

Determination of Phenolic Compounds Content
Phenolic compounds are often associated with various positive health effects, including antioxidant effects, decreases in the risk of cardiovascular diseases, anti-cancer mechanisms and anti-inflammatory properties [7,20].
The results of the total phenolic and total flavonoids content of E. globulus and C. ficifolia extracts are given in Table 2. The total phenolic content (TPC) was expressed as gallic acid equivalents (mg GAE/g dw plant material) and the calculation of total flavonoid content by using a standard curve of rutin and presented as rutin equivalents (mg RE/g dw plant material). The leaves of E. globulus contain an important amount of phenolic compounds (235.87 ± 4.38 mg/g dw plant material). The content of total phenolics in C. ficifolia was significantly lower (108.51 ± 1.43 mg/g dw plant material) but on the other hand higher, regarding the flavonoid content (44.44 ± 1.23 mg/g dw plant material). Regarding the total phenolics content of E. globulus leaves extract Pereira et al. reported a significantly lower amount of phenolic compounds (62.10 ± 2.49 mg/g dw plant material) in a 70% methanolic-aqueous extract [8]. Hassine et al. found a significantly lower amount of phenolics in an ethanolic extract of E. gillii (143.4 ± 0.1 mg/g dw plant material) but a comparable amount of flavonoids in the same extract (34.3 ± 0.1 mg/g dw plant material) [21]. The results of this study are also comparable with those of Luis et al. who found a total phenolic content of 218.67 ± 4.52 mg/g dw plant material in an 75% ethanolic wood extract of E. globulus [6]. No previous data were found regarding the total phenolics or flavonoid content in C. ficifolia extracts. The presence of biologically active compounds is regulated by a number of factors, including plant species, genetic factors, geographical location, type of soil, season of harvesting, herb preparation, drying and storage [22].

Antioxidant Activity Assays
The antioxidant potential of E. globulus and C. ficifolia extracts was tested through the DPPH bleaching method, Trolox equivalent antioxidant capacity (TEAC) assay, hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay and the inhibition of lipid peroxidation catalyzed by cytochrome c assay.
The results obtained by the DPPH assay are presented as quercetin equivalents (Table 3). According to this data, the E. glubulus leaves extract exhibited a higher antioxidant activity than C. ficifolia. In this case, the percentage of DPPH consumption was converted to quercetin equivalents by using a calibration curve (R 2 = 0.998) with quercetin solutions of 0-12 μM. The higher the rate of DPPH consumption is, the more powerful the antioxidant potential [22,23]. The TEAC results are in agreement with the DPPH values and also correlated with the results obtained by using the other methods. DPPH and TEAC assays are both based on the same principle (free radical scavenging by electron transfer mechanism) and use synthetic radicals which react directly with antioxidants to quantify the antioxidant capacity of the sample, the only notable difference is that in the case of the TEAC and HAPX, the solution is aqueous rather than ethanolic.
The enzymatic antioxidant assay (HAPX) measures the ability of the extract bioactive compounds to quench the damage inflicted by hydrogen peroxide upon hemoglobin. Additional information is provided by this assay, since it involves the interaction between antioxidant molecules with a protein, the ferryl hemoglobin species (resulting from the action of hydrogen peroxide on ferric hemoglobin) [24,25]. As one can see in the results of this study, the HAPX assay does not correlate with the DPPH and TEAC assays and indicates C. ficifolia extract as having a superior antioxidant activity. On the other hand, one can suppose that this result suggests a link between the higher content of flavonoids in C. ficifolia and the results from this assay.
A more physiological method based on peroxidase activity of cytochrome c was also assessed to evaluate the antioxidant activity of the extracts. This process monitors the formation of conjugated lipid dienes at 235 nm. According to Yang et al., the inhibition of lipid peroxidation increases with increasing concentrations of flavonoids [26]. Thus, the antioxidant capacity of the tested extracts, reflected in the delay of lipid oxidation, is considered to be based on the same mechanism found in HAPX: the interaction of antioxidants with ferryl hemoglobin, generated in this case by cytochrome c [25,27]. In this assay both extracts blocked the process of lipid peroxdation for the whole duration of the whole experiment (700 min), as can be seen in Figure 1.
The antioxidant potential E globulus and C. ficifolia leaves extracts was investigated using four different methods; the most frequent DPPH and TEAC assays and two complex and physiologically relevant methods based on peroxidase activity of hemoglobin and cytochrome c. As one can notice the antioxidant activity of vegetal extracts is strongly related with their chemical composition. As a peculiarity, these extracts contain important amounts of flavonoids and flavonols. High concentrations of flavonoids and flavonols are reflected in the significant scavenging properties [26,28]. There was a significant statistical difference between the analyzed extracts in the DPPH and HAPX assays (0.001 < p < 0.05) and a highly significant difference in TEAC method (p < 0.001).

Antimicrobial Activity Assays
Plants are important sources of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the assessment of their in vitro antibacterial activity [29]. The in vitro antibacterial potential of E. globulus and C. ficifolia extracts against both Gram-positive and Gram-negative bacteria is summarized in Table 4. As can be seen the E. globulus and C. ficifolia extracts showed important in vitro antibacterial activity against Staphylococcus aureus and Listeria monocytogenes. The results obtained in the present study revealed that C. ficifolia extract was more active than E. globulus against both Gram-positive and Gram-negative bacterial strains, with the exception of Bacillus subtilis. The best antibacterial activity was shown by both extracts against Listeria monocytogenes. The strain of Escherichia coli was also sensitive to the C. ficifolia extract with an inhibition zone of 7.3 ± 0.1 mm of diameter, higher that the inhibition zones of the two standard antibiotics under the present experimental conditions. The results obtained from the antimicrobial properties suggest that C. ficifolia is a source of antibiotics inhibiting microbial growth.
The MIC values obtained from antimicrobial tests ranged from 20 to >100 µg/mL ( Table 5). The results showed that the bacterial strain Staphylococcus aureus was the most sensitive to C. ficifolia extract, with a MIC value of 20 µg/mL. Listeria monocytogenes with a MIC value of 30 µg/mL was the most sensitive to E. globulus extract. Alternatively, Bacillus subtilis and Salmonella typhimurium were the least sensitive strains to both extracts, with MIC values >100 µg/mL. According to Salvat et al plant extracts with MICs of less than/or around 0.5 mg/mL indicate good antibacterial activity. Accordingly, E. globulus and C. ficifolia extracts exhibited good antimicrobial activity against most of the tested microorganisms [30]. Note: Each value is the mean ± SD of three independent measurements.

Plant Materials and Extraction Procedure
The vegetal material from E. globulus and C. ficifolia leaves was harvested from Perth National Park, Australia, in the summer of 2014. Voucher specimens (Voucher Nos. 390 and 391) were deposited in the Herbarium of the Environmental Science Department, Faculty of Environment Science and Engineering, Cluj-Napoca, Romania. The plant material was air dried at room temperature in the shade, separated and ground to a fine (≤300 µm) powder and then extracted. One gram of each sample was weighed and extracted with 10 mL of 70% ethanol, for 60 min in a sonicating bath at 60 °C. The samples were then cooled down and centrifuged at 4000 rpm for 30 min, and then the supernatant was recovered.

Apparatus and Chromatographic Conditions for the Analysis of Polyphenols
The identification and quantification of polyphenolic compounds was carried out using an Agilent Technologies 1100 HPLC Series system (Agilent, Santa Clara, CA, USA) equipped with G1322A degasser, G13311A binary gradient pump, column thermostat, G1313A autosampler and G1316A UV detector. The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). For the separation, a reverse-phase analytical column was employed (Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 μm particle) and the work temperature was set at 48 °C. The detection of the compounds was performed on both UV and MS mode. The UV detector was set at 330 nm until 17.5 min, then at 370 nm. The MS system operated using an electrospray ion source in the negative mode. ChemStation and DataAnalysis software from Agilent were used for processing the chromatographic data. The mobile phase was a binary gradient: methanol and acetic acid 0.1% (v/v). The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 min; then 42% methanol for the next 3 min [22,23,33]. The flow rate was 1 mL·min −1 and the injection volume was 5 µL.
The MS signal was used only for qualitative analysis based on the specific mass spectra of each polyphenol. The MS spectra obtained from a standard solution of polyphenols were integrated in a mass spectra library. Later, the MS traces/spectra of the analyzed samples were compared to spectra from library, which allows positive identification of compounds, based on spectral match. The UV trace was used for quantification of identified compounds from MS detection. Using the chromatographic conditions described above, the polyphenols eluted in less than 40 min. Four polyphenols could not be quantified in current chromatographic conditions due to overlapping (caftaric acid with gentisic acid and caffeic acid with chlorogenic acid). However, all four compounds could be selectively identified in MS detection (qualitative analysis) based on differences between their pseudo-molecular mass and MS spectra. For all compounds, the limit of quantification was 0.5 μg/mL, and the limit of detection was 0.1 μg/mL. The detection limits were calculated as minimal concentration producing a reproductive peak with a signal-to-noise ratio greater than three. Quantitative determinations were performed using an external standard method. Calibration curves in the 0.5-50 μg/mL range with good linearity (R 2 > 0.999) for a five point plot were used to determine the concentration of polyphenols in plant samples [22,23,33,34].

Identification and Quantification of Polyphenols
The detection and quantification of polyphenols was performed in UV-assisted mass spectrometry detection. Due to peak overlapping, four polyphenol-carboxylic acids (caftaric, gentisic, caffeic, chlorogenic) were determined only based on MS spectra, whereas for the rest of the compounds the linearity of the calibration curves was very good (R 2 > 0.998), with detection limits in the range of 18 ng/mL to 92 ng/mL. The detection limits were calculated as the minimal concentration yielding a reproductible peak with a signal-to-noise ratio greater than three. Quantitative determinations were performed using an external standard method; retention times were determined with a standard deviation ranging from 0.04 min to 0.19 min. For all compounds, the accuracy was between 94.13% and 105.3%. Accuracy was checked by spiking samples with a solution containing each polyphenol in a 10 μg/mL concentration. In all analyzed samples the compounds were identified by comparison of their retention times and recorded electrospray mass spectra with those of standards in the same chromatographic conditions.

Determination of Total Polyphenols and Flavonoids Content
The total phenolic content (TPC) of the extracts was measured using the Folin-Ciocalteu method with some modifications [23,34]. Two mL from each ethanolic extract were diluted 25 times and then mixed with Folin-Ciocalteu reagent (1 mL) and distilled water (10.0 mL) and diluted to 25.0 mL with a 290 g/L solution of sodium carbonate. The samples were incubated in the dark for 30 min. The absorbance was measured at 760 nm, using a JASCO UV-VIS spectrophotometer. Standard curve was prepared by using different concentrations of gallic acid and the absorbances were measured at 760 nm. TPC values were determined using an equation obtained from the calibration curve of gallic acid graph (R 2 = 0.999). Total polyphenolic content was expressed as mg gallic acid/g dry material plant (mg GAE/g plant material).
The total flavonoids content was calculated and expressed as rutin equivalents after the method described in the Romanian Pharmacopoeia (Xth Edition) [35]. Each extract (5 mL) was mixed with sodium acetate (5.0 mL, 100 g/L), aluminum chloride (3.0 mL, 25 g/L), and made up to 25 mL in a calibrated flask with methanol. Each solution was compared with the same mixture without reagent. The absorbance was measured at 430 nm. The total flavonoids content values were determined using an equation obtained from calibration curve of the rutin graph (R 2 = 0.999).

DPPH Bleaching Assay
The DPPH assay provides an easy and rapid way to evaluate potential antioxidants. DPPH free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in ethanol. This free radical, stable at room temperature is reduced in the presence of an antioxidant molecule, giving rise to a yellow solution. The free radical scavenging activity of the ethanolic extracts was measured in terms of hydrogen donating or radical scavenging ability using this method. A stock solution of 100 µM DPPH was prepared. In a glass cuvette, 2 µL from original extracts were added to 998 µL DPPH solution. The absorbance changes were monitored at 517 nm for 30 min, using a UV-VIS spectrophotometer equipped with a multi-cell holder. The percentage of DPPH consumption in each case was converted to quercetin equivalents using a calibration curve (R 2 = 0.991) with quercetin standard solutions of 0-12 µM [23,34]. The higher the rate of DPPH consumption, the more powerful is the antioxidant capacity.

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
In the Trolox equivalent antioxidant capacity (TEAC) assay, the antioxidant capacity is reflected in the ability of the natural extracts to decrease the color, reacting directly with the ABTS radical. The latter was obtained by oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with peroxide, catalyzed by horseradish peroxidase (HRP). Original extracts were diluted 5 times, and 3 µL from the diluted extract were added to 997 µL ABTS solution. The amount of ABTS radical consumed by the tested compound was measured at 735 nm, after 30 min of reaction time. The evaluation of the antioxidant capacity was obtained using the total change in absorbance at this wavelength. The percentage of ABTS consumption was transformed in Trolox equivalents (TE) using a calibration curve (R 2 = 0.986) with Trolox standard solutions of 0-16 µM [22,23].

Hemoglobin/Ascorbate Peroxidase Activity Inhibition (HAPX) Assay
Inhibition of hemoglobin ascorbate peroxidase activity assay (HAPX) was conducted according to the procedure described by Mot et al. [24]. Hemoglobin was purified according to the Antonini and Brunori protocols [31]. The reaction was triggered by the addition of met hemoglobin (6 µM) to a mixture of ascorbate (160 µM), peroxide (700 µM) and extracts (5 µM) from the stock solution diluted five times, and it was monitored at 405 nm. This method allows us to evaluate the inhibition of ferryl formation by ascorbate in the presence of the tested compounds. An increase in the time of inhibition reflects the antioxidant capacity of the compound, whereas a decrease, indicates a pro-oxidant effect [32].

Inhibition of Lipid Peroxidation Catalyzed by Cytochrome c
Liposomes were obtained by suspending 5 mg/mL soybean lecithin in phosphate buffer (20 mM, pH 7), followed by sonication for 15 min in an ultrasonic bath (using a Power Sonic 410 device, Thermoline Scientific, Wetherill Park, NSW, Australia). The liposome oxidation experiment was performed at room temperature, for 700 min, in the presence of cytochrome c (2 µM) and extracts (5 µL from the diluted extract) by monitoring the absorbance at 235 nm (wavelength specific for liposome oxidation). This process monitors the formation of lipid conjugated dienes at the specified wavelength [25].

Microorganisms and Culture Growth
The microorganisms used for antimicrobial activity evaluation were obtained from the University of Agricultural Sciences and Veterinary Medicine Cluj Napoca, Romania. The Gram-positive bacteria Staphylococcus aureus (ATCC-25923), Bacillus subtilis (ATCC-12228), Listeria monocytogenes (ATCC-19115) and gram-negative bacteria Escherichia coli (ATCC-25922) and Salmonella typhimurium (ATCC-14028). The stock cultures of microorganisms used in this study were maintained on plate count agar slants at 4 °C. Inoculum was prepared by suspending a loop full of bacterial cultures into 10 mL of nutrient agar broth and was incubated at 37 °C for 24 h. About 60 µL of bacterial suspensions, adjusted to 10 6 CFU/mL were taken and poured into Petri plates containing 10 mL sterilized nutrient agar medium. Bacterial suspensions were spread to get a uniform lawn culture [36].

Antimicrobial Activity Assay
Antimicrobial activities of the E. globulus and C. ficifolia extracts were evaluated by means of agar-well diffusion assay with some modifications [36]. Fifteen millilitres of the molten agar (45 °C) were poured into sterile Petri dishes (Ø 90 mm). Cell suspensions were prepared and 100 µL was evenly spreader onto the surface of the agar plates of Mueller-Hinton agar (Oxoid, Basingstoke, UK). Once the plates had been aseptically dried, 6 mm wells were punched into the agar with a sterile Pasteur pipette. The different extracts (10 mg/mL) were dissolved in dimethylsulfoxide/water (1/9) and 80 µL were placed into the wells and the plates were incubated at 37 °C for 24 h. Gentamicin (25 µL/wells at concentration of 4 µg/mL) and ciprofloxacin (5 µg/mL) were used as positive control for bacteria. Antimicrobial activity was evaluated by measuring the diameter of circular inhibition zones around the well. Tests were performed in triplicate and values are the averages of three replicates [30].

Minimum Inhibitory Concentration
Based on the previous screening the minimum inhibitory concentration (MIC) of both E. globulus and C. ficifolia extracts was analyzed through the agar-well diffusion method. A bacterial suspension (10 5 -10 6 CFU/mL) of each tested microorganism was spread on the nutrient agar plate. The wells (6 mm diameter) were cut from agar, and 60 µL of each E. globulus and C. ficifolia extracts dissolved in dimethyl sulfoxide (DMSO) at different concentrations (10,20,25, 50 75 and 100 µg/mL) were delivered into them. The plates were incubated at 37 °C for 24 h under aerobic conditions that followed by the measurement of the diameter of the inhibition zone expressed in millimeter. MIC was taken from the concentration of the lowest dosed well visually showing no growth after 24 h [30].

Statistical Analysis
The experiments were designed and the experimental data evaluated using one-way analysis of variance (ANOVA), with p < 0.05 as threshold for statistical significance. The statistical results confirm the hypothesis that the differences between the results are either not significant (p > 0.05), significant (0.001 < p < 0.05) or highly significant (p < 0.001). The average of multiple measurements (triplicates or more) was listed in the tables together with the standard deviations. Statistical analysis was performed using Excel software package.

Conclusions
The results of the present study reveal important data regarding the phenolic composition, antioxidant and antibacterial activities of the two medicinal species E. globulus and C. ficifolia. The differences between the two species are mostly quantitative and are related to the respective flavonoid and flavonol profiles. The dominant flavonoid glycoside in both species is hyperoside, with its highest amount being found in E. globulus. Regarding the flavonol profile, C. ficifolia contains higher amounts in each of the identified compounds. Nevertheless gentisic and p-coumaric acids were found just in the C. ficifolia extract. The antioxidant potential of the two species was tested through several assays, which indicated both species as valuable sources of free radical scavenging compounds. However, the HAPX assay results don't correlate with the DPPH and TEAC assays and show C. ficifolia extract as having a superior antioxidant activity. This might suggest a link between the higher content of flavonoids in C. ficifolia and the results from this assay. Referring to antibacterial assays, C. ficifolia extract was found to be more active than E. globulus against both Gram-positive and Gram-negative bacterial strains with the exception of Bacillus subtilis; the best antibacterial activity was shown against Listeria monocytogenes. The MIC results showed that the bacterial strain Staphylococcus aureus was the most sensitive to C. ficifolia extract and Listeria monocytogenes was the most sensitive to E. globulus extract.