Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Citrus limon Essential Oil: Chemical Composition and Selected Biological Properties Focusing on the Antimicrobial (In Vitro, In Situ), Antibiofilm, Insecticidal Activity and Preservative Effect against Salmonella enterica Inoculated in Carrot.

New goals for industry and science have led to increased awareness of food safety and healthier living in the modern era. Here, one of the challenges in food quality assurance is the presence of pathogenic microorganisms. As planktonic cells can form biofilms and go into a sessile state, microorganisms are now more resistant to broad-spectrum antibiotics. Due to their proven antibacterial properties, essential oils represent a potential option to prevent food spoilage in the search for effective natural preservatives. In this study, the chemical profile of Citrus limon essential oil (CLEO) was evaluated. GC-MS analysis revealed that limonene (60.7%), ß-pinene (12.6%), and γ-terpinene (10.3%) are common constituents of CLEO, which prompted further research on antibacterial and antibiofilm properties. Minimum inhibitory concentration (MIC) values showed that CLEO generally exhibits acceptable antibacterial properties. In addition, in situ antimicrobial research revealed that vapour-phase CLEO can arrest the growth of Candida and Y. enterocolitica species on specific food models, indicating the potential of CLEO as a preservative. The antibiofilm properties of CLEO were evaluated by MIC assays, crystal violet assays, and MALDI-TOF MS analysis against S. enterica biofilm. The results of the MIC and crystal violet assays showed that CLEO has strong antibiofilm activity. In addition, the data obtained by MALDI-TOF MS investigation showed that CLEO altered the protein profiles of the bacteria studied on glass and stainless-steel surfaces. Our study also found a positive antimicrobial effect of CLEO against S. enterica. The anti-Salmonella activity of CLEO in vacuum-packed sous vide carrot samples was slightly stronger than in controls. These results highlight the advantages of the antibacterial and antibiofilm properties of CLEO, suggesting potential applications in food preservation.

Anti-Salmonella Activity of Thymus serpyllum Essential Oil in Sous Vide Cook-Chill Rabbit Meat.

Food is generally prepared and vacuum-sealed in a water bath, then heated to a precise temperature and circulated in a sous vide machine. Due to its affordability and ease of use, this cooking method is becoming increasingly popular in homes and food service businesses. However, suggestions from manufacturers and chefs for long-term, low-temperature sous vide cooking raise questions about food safety in the media. In this study, heat treatment with different times and wild thyme essential oil (EO) in sous vide-processed rabbit longissimus dorsi muscle were found to inactivate Salmonella enterica. The rabbit meat samples were vacuum-packed in control groups, in the second group the rabbit meat samples were injected with S. enterica, and in the third group were meat samples infected with S. enterica with Thymus serpylum EO additive. The vacuum-packed samples were cooked sous vide for the prescribed time at 55, 60, and 65 °C. At 5, 15, 30, and 60 min, the quantities of S. enterica, total bacterial counts, and coliform bacteria were measured in groups of sous vide rabbit meat. Microbiological analyses of rabbit meat samples on days 1 and 7 were evaluated. In this study, total viable counts, coliforms bacteria, and number of Salmonella spp. were identified. After incubation, isolates from different groups of microorganisms were identified by the mass spectrometry technique. For each day measured, the test group exposed to a temperature of 55 °C for 5 min had a greater number of total microbiota. The most isolated microorganisms by MALDI-TOF MS Biotyper from the control and treated groups were Lactococcus garvieae and in the treated groups also S. enterica. Based on our analysis of sous vide rabbit meat samples, we discovered that adding 1% of thyme essential oil to the mixture reduced the amount of Salmonella cells and increased the overall and coliform bacterial counts. The microbiological quality of sous vide rabbit meat that was kept for seven days was positively impacted by the addition of thyme essential oil.

Thymus zygis, Valuable Antimicrobial (In Vitro and In Situ) and Antibiofilm Agent with Potential Antiproliferative Effects.

With the growing issues of food spoilage, microbial resistance, and high mortality caused by cancer, the aim of this study was to evaluate T. zygis essential oil (TZEO) as a potential solution for these challenges. Here, we first performed GC/MS analysis which showed that the tested TZEO belongs to the linalool chemotype since the abundance of linalool was found to be 38.0%. Antioxidant activity assays showed the superiority of TZEO in neutralizing the ABTS radical cation compared to the DPPH radical. The TZEO was able to neutralize 50% of ABTS•+ at the concentration of 53.03 ± 1.34 µg/mL. Antimicrobial assessment performed by employing disc diffusion and minimal inhibitory concentration assays revealed TZEO as a potent antimicrobial agent with the highest inhibition activity towards tested gram-negative strains. The most sensitive on the treatment with TZEO was Enterobacter aerogenes showing an MIC 50 value of 0.147 ± 0.006 mg/mL and a MIC 90 value of 0.158 ± 0.024 mg/mL. Additionally, an in situ analysis showed great effects of TZEO in inhibiting gram-negative E. coli, P. putida, and E. aerogenes growing on bananas and cucumbers. Treatment with the TZEO vapor phase in the concentration of 500 µg/mL was able to reduce the growth of these bacteria on the food models to the extent > 90%, except for E. coli growth on the cucumber, which was reduced to the extent of 83.87 ± 4.76%. Furthermore, a test on the antibiofilm activity of the tested essential oil revealed its biofilm prevention effects against Salmonella enterica which forms biofilms on plastic and stainless-steel surfaces. Performed tests on the TZEO effects towards cell viability showed no effects on the normal MRC-5 cell line. However, the results of MTT assay of TZEO effects on three cancer cell lines (MDA-MB-231, HCT-116, and K562) suggest that TZEO exerted the strongest effects on the inhibition of the viability of MDA-MB-231 cells, especially after long-term treatment in the highest concentration applied with reducing the viability of the cells to 57%. Additionally, results of NBT and Griess assays suggest that TZEO could be a convenient candidate for future testing for developing novel antitumor therapies.

Sage Essential Oil as an Antimicrobial Agent against Salmonella enterica during Beef Sous Vide Storage.

Sous-vide is a process comprising vacuum-sealing food, heating it to the desired temperature, and circulating it in a water bath in a sous vide machine. This cooking technique is increasingly common in homes and catering establishments due to its simplicity and affordability. However, manufacturers and chef's recommendations for low-temperature and long-term sous-vide cooking in media raise food safety concerns, particularly when preparing beef tenderloin. In this study, Salmonella enterica was found to be inactivated by heat and sage essential oil (EO) in beef samples from musculus psoas major that had been sous vide processed. To determine whether heat treatment was likely to increase the sous vide efficiency, S. enterica and sage EO were mixed. After being vacuum-packed and injected with S. enterica, the samples were cooked at 50-65 °C through the sous vide technique for the prescribed time. On days 1, 3, and 6, the amounts of S. enterica, total bacteria, and coliform bacteria were measured in the control and treated groups of beef processed sous vide. Mass spectrometry was used to identify bacterial isolates on different days. On each day that was measured, a higher number of all the microbiota was found in the samples exposed to 50 °C for 5 min. The most frequently isolated microorganisms from both groups of samples were Pseudomonas fragi (17%), Pseudomonas cedrina (8%), and Proteus vulgaris (8%); in the treated group, also S. enterica (21%), Pseudomonas fragi (13%), and Pseudomonas veronii (6%). After the heat treatment of samples at 65 °C for 20 min, the total count of bacteria and coliform bacteria was zero. It has been shown that adding sage essential oil (EO) in combination with sous vide processing technique leads to the stabilization and safety of beef tenderloin.

Antilisterial and Antimicrobial Effect of Salvia officinalis Essential Oil in Beef Sous-Vide Meat during Storage.

If food is contaminated with pathogens such as Listeria monocytogenes, improper cooking during sous-vide preparation can lead to foodborne illnesses. In this study, it was found that L. monocytogenes were inactivated with both heat and the essential oil of Salvia officinalis (sage EO) in beef tenderloin of the musculus psoas major that had undergone sous-vide processing. To determine whether the enhancement of the efficacy of heat treatment is prospective, L. monocytogenes and sage EO were mixed. Groups with L. monocytogenes alone and sage essential oil combined with L. monocytogenes and test groups without EO were established. The samples were vacuum-packed, inoculated with L. monocytogenes, and then cooked sous-vide for the predetermined duration at 50, 55, 60, or 65 °C. In both groups with sous-vide beef tenderloin, the total bacterial count, the coliforms bacterial count, and the amount of L. monocytogenes were assessed on days 0, 3, 6, 9, and 12. Over these days, the amounts of L. monocytogenes, coliform bacteria, and overall bacteria increased. The identification of bacterial strains in various days and categories was performed by MALDI-TOF mass spectrometry. The test group that was exposed to a temperature of 50 °C for 5 min had a higher overall bacterial count for each day that was assessed. Pseudomonas fragi and L. monocytogenes were the most isolated organisms from the test group and the treated group. To ensure the safety for the consumption of sous-vide beef tenderloin, it was found that the addition of natural antimicrobials could produce effective outcomes.

Salvia sclarea Essential Oil Chemical Composition and Biological Activities.

Salvia sclarea essential oil (SSEO) has a long tradition in the food, cosmetic, and perfume industries. The present study aimed to analyze the chemical composition of SSEO, its antioxidant activity, antimicrobial activity in vitro and in situ, antibiofilm, and insecticidal activity. Besides that, in this study, we have evaluated the antimicrobial activity of SSEO constituent (E)-caryophyllene and standard antibiotic meropenem. Identification of volatile constituents was performed by using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) techniques. Results obtained indicated that the main constituents of SSEO were linalool acetate (49.1%) and linalool (20.6%), followed by (E)-caryophyllene (5.1%), p-cimene (4.9%), a-terpineol (4.9%), and geranyl acetate (4.4%). Antioxidant activity was determined as low by the means of neutralization of the DDPH radical and ABTS radical cation. The SSEO was able to neutralize the DPPH radical to an extent of 11.76 ± 1.34%, while its ability to decolorize the ABTS radical cation was determined at 29.70 ± 1.45%. Preliminary results of antimicrobial activity were obtained with the disc diffusion method, while further results were obtained by broth microdilution and the vapor phase method. Overall, the results of antimicrobial testing of SSEO, (E)-caryophyllene, and meropenem, were moderate. However, the lowest MIC values, determined in the range of 0.22-0.75 µg/mL for MIC50 and 0.39-0.89 µg/mL for MIC90, were observed for (E)-caryophyllene. The antimicrobial activity of the vapor phase of SSEO (towards microorganisms growing on potato) was significantly stronger than that of the contact application. Biofilm analysis using the MALDI TOF MS Biotyper showed changes in the protein profile of Pseudomonas fluorescens that showed the efficiency of SSEO in inhibiting biofilm formation on stainless-steel and plastic surfaces. The insecticidal potential of SSEO against Oxycarenus lavatera was also demonstrated, and results show that the highest concentration was the most effective, showing insecticidal activity of 66.66%. The results obtained in this study indicate the potential application of SSEO as a biofilm control agent, in the shelf-life extension and storage of potatoes, and as an insecticidal agent.

Chemical Composition and Biological Activities of Eucalyptus globulus Essential Oil.

Eucalyptus globulus essential oil (EGEO) is considered as a potential source of bioactive compounds with significant biological activity. The aim of this study was to analyze the chemical composition of EGEO, in vitro and in situ antimicrobial activity, antibiofilm activity, antioxidant activity, and insecticidal activity. The chemical composition was identified using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The main components of EGEO were 1,8-cineole (63.1%), p-cimene (7.7%), a-pinene (7.3%), and a-limonene (6.9%). Up to 99.2% of monoterpenes were present. The antioxidant potential of essential oil and results indicate that 10 µL of this sample can neutralize 55.44 ± 0.99% of ABTS•+, which is equivalent to 3.22 ± 0.01 TEAC. Antimicrobial activity was determined via two methods: disk diffusion and minimum inhibitory concentration. The best antimicrobial activity was shown against C. albicans (14.00 ± 1.00 mm) and microscopic fungi (11.00 ± 0.00 mm-12.33 ± 0.58 mm). The minimum inhibitory concentration showed the best results against C. tropicalis (MIC 50 2.93 µL/mL, MIC 90 3.17 µL/mL). The antibiofilm activity of EGEO against biofilm-forming P. flourescens was also confirmed in this study. The antimicrobial activity in situ, i.e., in the vapor phase, was significantly stronger than in the contact application. Insecticidal activity was also tested and at concentrations of 100%, 50%, and 25%; the EGEO killed 100% of O. lavaterae individuals. EGEO was comprehensively investigated in this study and information regarding the biological activities and chemical composition of the essential oil of Eucalyptus globulus was expanded.

Biological Activity of Cupressus sempervirens Essential Oil.

The aim of this study was to evaluate the antioxidant, antibiofilm, antimicrobial (in situ and in vitro), insecticidal, and antiproliferative activity of Cupressus sempervirens essential oil (CSEO) obtained from the plant leaf. The identification of the constituents contained in CSEO was also intended by using GC and GC/MS analysis. The chemical composition revealed that this sample was dominated by monoterpene hydrocarbons α-pinene, and δ-3-carene. Free radical scavenging ability, performed by using DPPH and ABTS assays, was evaluated as strong. Higher antibacterial efficacy was demonstrated for the agar diffusion method compared to the disk diffusion method. The antifungal activity of CSEO was moderate. When the minimum inhibitory concentrations of filamentous microscopic fungi were determined, we observed the efficacy depending on the concentration used, except for B. cinerea where the efficacy of lower concentration was more pronounced. The vapor phase effect was more pronounced at lower concentrations in most cases. Antibiofilm effect against Salmonella enterica was demonstrated. The relatively strong insecticidal activity was demonstrated with an LC50 value of 21.07% and an LC90 value of 78.21%, making CSEO potentially adequate in the control of agricultural insect pests. Results of cell viability testing showed no effects on the normal MRC-5 cell line, and antiproliferative effects towards MDA-MB-231, HCT-116, JEG-3, and K562 cells, whereas K562 cells were the most sensitive. Based on our results, CSEO could be a suitable alternative against different types of microorganisms as well as suitable for the control of biofilms. Due to its insecticidal properties, it could be used in the control of agricultural insect pests.

An In-Depth Study on the Chemical Composition and Biological Effects of Pelargonium graveolens Essential Oil.

The essential oil of Pelargonium graveolens (PGEO) is identified in the literature as a rich source of bioactive compounds with a high level of biological activity. This study aimed to examine the chemical profile of PGEO as well as its antioxidant, antibacterial, antibiofilm, and insecticidal properties. Its chemical composition was analyzed using gas chromatography-mass spectrometry (GC-MS), achieving comprehensive identification of 99.2% of volatile compounds. The predominant identified compounds were ß-citronellol (29.7%) and geraniol (14.6%). PGEO's antioxidant potential was determined by means of DPPH radical and ABTS radical cation neutralization. The results indicate a higher capacity of PGEO to neutralize the ABTS radical cation, with an IC50 value of 0.26 ± 0.02 mg/mL. Two techniques were used to assess antimicrobial activity: minimum inhibitory concentration (MIC) and disk diffusion. Antimicrobial evaluation using the disk diffusion method revealed that Salmonella enterica (14.33 ± 0.58 mm), which forms biofilms, and Priestia megaterium (14.67 ± 0.58 mm) were most susceptible to exposure to PGEO. The MIC assay demonstrated the highest performance of this EO against biofilm-forming S. enterica (MIC 50 0.57 ± 0.006; MIC 90 0.169 ± 0.08 mg/mL). In contrast to contact application, the assessment of the in situ vapor phase antibacterial activity of PGEO revealed significantly more potent effects. An analysis of antibiofilm activity using MALDI-TOF MS demonstrated PGEO's capacity to disrupt the biofilm homeostasis of S. enterica growing on plastic and stainless steel. Additionally, insecticidal evaluations indicated that treatment with PGEO at doses of 100% and 50% resulted in the complete mortality of all Harmonia axyridis individuals.

Chemical Composition and Biological Activity of Tanacetum balsamita Essential Oils Obtained from Different Plant Organs.

The aim of this study is to evaluate the chemical composition of Tanacetum balsamita L. essential oils (EOs) obtained from different plant organs, flowers (FEO), leaves (LEO), and stems (SEO), as well as to assess their biological properties. The results obtained by using GC and GC/MS analysis indicate that this plant belongs to the carvone chemotype. Moreover, we examined the oil's antimicrobial and antitumor potential. Antimicrobial effects were determined using minimum inhibitory concentrations assay and the vapor phase method. Obtained results indicate better antimicrobial activity of investigated EO samples compared to the commercially available antibiotics. On the treatment with FEO, Y. enterocolitica and H. influenzae showed high sensitivity, while treatment with LEO and SEO showed the highest effects against S. aureus. The vapor phase method, as an in situ antibacterial analysis, was performed using LEO. Obtained results showed that this EO has significant activity toward S. pneumoniae in the apple and carrot models, L. monocytogenes in the pear model, and Y. enterocolitica in the white radish model. The potential antitumor mechanisms of FEO, LEO, and SEO were determined by the means of cell viability, redox potential, and migratory capacity in the MDA-MB-231 and MDA-MB-468 cell lines. The results show that these EOs exert antiviability potential in a time- and dose-dependent manner. Moreover, treatments with these EOs decreased the levels of superoxide anion radical and increased the levels of nitric oxide in both tested cell lines. The results regarding total and reduced glutathione revealed, overall, an increase in the levels of total glutathione and a decrease in the levels of reduced glutathione, indicating strong antioxidative potential in tested cancer cells in response to the prooxidative effects of the tested EOs. The tested EOs also exerted a drop in migratory capacity, which indicates that they can be potentially used as chemotherapeutic agents.

Chemical Composition, Antioxidant, In Vitro and In Situ Antimicrobial, Antibiofilm, and Anti-Insect Activity of Cedar atlantica Essential Oil.

The present study was designed to evaluate commercial cedar essential oil (CEO), obtained by hydrodistillation from cedar wood, in relationship to its chemical composition and antioxidant, in vitro and in situ antimicrobial, antibiofilm, and anti-insect activity. For these purposes, gas chromatography-mass spectrometry, DPPH radical-scavenging assay, agar and disc diffusion, and vapor phase methods were used. The results from the volatile profile determination showed that δ-cadinene (36.3%), (Z)-ß-farnesene (13.8%), viridiflorol (7.3%), and himachala-2,4-diene (5.4%) were the major components of the EO chemical constitution. Based on the obtained results, a strong antioxidant effect (81.1%) of the CEO was found. CEO is characterized by diversified antimicrobial activity, and the zones of inhibition ranged from 7.33 to 21.36 mm in gram-positive and gram-negative bacteria, and from 5.44 to 13.67 mm in yeasts and fungi. The lowest values of minimal inhibition concentration (MIC) were noted against gram-positive Micrococcus luteus (7.46 µL/mL) and against yeast Candida krusei (9.46 µL/mL). It seems that the vapor phase of CEO can inhibit the growth of the microscopic filamentous fungi of the genus Penicillium according to in situ antifungal analysis on bread, carrots, and celery. This finding confirms the impact of CEO on the change in the protein structure of older biofilms of Pseudomonas fluorescens and Salmonella enterica subsp. enterica. Insecticidal activity of a vapor phase has also been demonstrated against Pyrrhocoris apterus. CEO showed various advantages on antimicrobial activity, and it is an ideal substitute for food safety.

Thymus vulgaris Essential Oil and Its Biological Activity.

Thymus vulgaris essential oil has potential good biological activity. The aim of the research was to evaluate the biological activity of the T. vulgaris essential oil from the Slovak company. The main components of T. vulgaris essential oil were thymol (48.1%), p-cymene (11.7%), 1,8-cineole (6.7), γ-terpinene (6.1%), and carvacrol (5.5%). The antioxidant activity was 85.2 ± 0.2%, which corresponds to 479.34 ± 1.1 TEAC. The antimicrobial activity was moderate or very strong with inhibition zones from 9.89 to 22.44 mm. The lowest values of MIC were determined against B. subtilis, E. faecalis, and S. aureus. In situ antifungal analysis on bread shows that the vapor phase of T. vulgaris essential oil can inhibit the growth of the microscopic filamentous fungi of the genus Penicillium. The antimicrobial activity against S. marcescens showed 46.78-87.80% inhibition at concentrations 62.5-500 µL/mL. The MALDI TOF MS analyses suggest changes in the protein profile of biofilm forming bacteria P. fluorescens and S. enteritidis after the fifth and the ninth day, respectively. Due to the properties of the T. vulgaris essential oil, it can be used in the food industry as a natural supplement to extend the shelf life of the foods.