The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.

Occurrence of Hydroxytyrosol, Tyrosol and Their Metabolites in Italian Cheese.

Tyrosol (T) and hydroxytyrosol (HT) are phenyl alcohol polyphenols with well-recognized health-promoting properties. They are widely diffused in several vegetables, especially in olive products (leaves, fruits and oil). Therefore, they could be present in food produced from herbivorous animals such as in milk and cheese. In this study, an analytical method to determine T, HT and some of their phase II metabolites (sulphates and glucuronides) in cheese was developed and validated. Samples were extracted with an acidic mixture of MeOH/water 80/20 (v/v) and, after a low temperature clean-up, the extracts were evaporated and injected in a liquid-chromatography coupled with high resolution mass spectrometry (LC-Q-Orbitrap). A validation study demonstrated satisfactory method performance characteristics (selectivity, linearity, precision, recovery factors, detection and quantification limits). The developed protocol was then applied to analyze 36 Italian cheeses made from ewe, goat and cow milk. The sum of detected compounds (T, tyrosol sulfate, hydroxytyrosol-3-O-sulfate and hydroxytyrosol-4-O-sulfate) reached as high as 2300 µg kg-1 on a dry weight basis, although in about 45% of cow cheeses it did not exceed 50 µg kg-1. Ewe cheeses were significantly richer of polyphenols (sum) as well as HT sulfate metabolites than cow cheeses. In conclusion, results shows that cheese cannot be considered an important dietary source of these valuable compounds.

Effect of the Application of a Green Preservative Strategy on Minced Meat Products: Antimicrobial Efficacy of Olive Mill Wastewater Polyphenolic Extract in Improving Beef Burger Shelf-Life.

The mincing process of raw meat favors microbial spoilage as well as chemical and enzymatic oxidation processes. In order to limit this degradative process, preservatives are routinely added to minced meat products. The role of olive mill wastewater polyphenolic extract as a replacement for synthetic preservatives in beef burger was assessed. The antioxidant capacity of the extract experimentally added to beef burger was evaluated using the oxygen radical absorbance capacity method (ORACFL) to assess the shelf-life, while the lipid oxidation was measured by thiobarbituric reactive substance (TBAR) determination. The antimicrobial activity was assayed by means of classical methods and predictive microbiology. The experimental addition of polyphenolic extract led to 62% lower lipid oxidation and 58% higher antioxidant capacity; it also successfully modulated spoilage microbial populations with an average growth reduction of 15% on day 7. Results indicate that olive mill wastewater polyphenolic extracts could be added to raw ground beef meat to act as natural antioxidants and to modulate microbial growth.

Antibacterial Activity of Crocus sativus L. Petals Extracts against Foodborne Pathogenic and Spoilage Microorganisms, with a Special Focus on Clostridia.

In recent years, there has been a growing interest in the use of novel antimicrobial agents able to inhibit or kill food-borne bacteria or to interrupt the onset of food spoilage. Crocus sativus L. petals, typically considered as waste obtained from saffron spice production, could be a source of natural bioactive compounds to be used as food preservatives. The purpose of this work was to investigate the antibacterial properties of two hydroalcoholicsaffron petal extracts obtained by maceration (SPEA) and by ultrasonic bath (SPEB) methods. The main polyphenols identified in both extracts were gallic and chlorogenic acids, representing almost 70% of the phenolic fraction monitored. The antibacterial activity was studied by the agar well-diffusion method, against food-borne pathogenic and spoilage bacteria. Both extracts showed activity mainly against Gram-positive bacteria, in particular those belonging to the Clostridiaceae family (C. perfringens, C. botulinum and C. difficile), with inhibition zone diameters ranging from 13 to 18 mm. The antibacterial properties against Clostridia were further analyzed, determining MIC and MBC and performing a time-kill test. SPEA showed lower MIC/MBC values (250 mg/mL) compared to SPEB (500 mg/mL), suggesting that it could be more active against the assayed strains, probably because of its higher content of gallic acid. SPEA and SPEB, tested at a concentration of 1 × MIC, showed bactericidal activity against C. perfringens, C. botulinum and C. difficile and these results suggest that saffron petals could represent a valuable natural alternative source to conventional preservatives. Further investigations are needed to evaluate possible applications in the food industry.

Dietary Supplementation with Olive Mill Wastewater in Dairy Sheep: Evaluation of Cheese Characteristics and Presence of Bioactive Molecules.

The aim of the study was to define the chemical characteristics, antioxidant capacity, oxidative status, sensory properties, and the presence of polyphenols in ovine cheese obtained after dietary administration of spray-dried olive mill wastewater (SDP). SDP is a waste from olive oil production rich in bioactive molecules obtained by further processing the olive mill wastewater through a spray-drying system. Thirty-six sheep were randomly assigned to two experimental groups that received a standard diet based on hay and concentrate. The concentrate fed to the SDP group was supplemented with SDP at a rate 25 g/kg (as fed). The trial lasted 9 weeks. Milk from the two treatment groups was separately collected and used for manufacturing cheese. Cheese quality parameters and proximate composition were not affected by the dietary treatment, whereas the antioxidant status and oxidative stability of cheese were positively affected. Polyphenol analyses in cheese were performed through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The concentration of hydroxytyrosol and tyrosol, and their sulphate metabolites, were higher in cheese from supplemented sheep. These findings suggest that polyphenol metabolites can play a major role in the beneficial effects observed in food produced from sheep fed SDP.

Antimicrobial and anti-biofilm activity of olive oil by-products against Campylobacter spp. isolated from chicken meat.

BACKGROUND: Worldwide, poultry is considered the main source of food-related human campylobacteriosis, which is generally associated with the consumption of raw or undercooked chicken meat. Furthermore, Cam- pylobacter develops biofilms that are resistant to environmental stress, antibiotics, and disinfectants and are becoming a major issue for the food industry, especially the poultry industry. This study investigated the antimicrobial and anti-biofilm properties of polyphenols found in spray-dried olive mill wastewater (OMWW--SD) against Campylobacter strains isolated from chicken meat. METHODS: OMWW-SD was produced by dehydration of olive mill wastewater polyphenolic extract. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for OMWW-SD were determined by microdilution method whereas the inhibitory effect of the OMWW-SD on biofilm formation and biofilm disaggregation was tested through crystal violet assay on polystyrene plates. RESULTS: The phenolic profile of OMWW-SD mainly consisted of secoiridoid and hydroxycinnamic acid derivatives. Oleuropein-aglycone di-aldehyde (a secoiridoid derivative) was the major constituent, representing 72.5% of the total identified phenolic compounds. OMWW-SD showed a MIC ranging from 0.15 mg/mL to 0.3 mg/mL and a MBC of 0.3 mg/mL for all Campylobacter strains tested. The olive by-product extract tested was able, in vitro, to inhibit biofilm formation and to promote biofilm dispersion even at sub-MIC concentra- tions, with values ranging from 6% to 92% and from 4% to 83% at varying extract dilutions, respectively. CONCLUSIONS: OMWW-SD could be developed as a new anti-biofilm agent with potential to control Campylo- bacter in the food chain, especially in the poultry industry, thereby enhancing food safety.

Antimicrobial efficacy of a polyphenolic extract from olive oil by-product against "Fior di latte" cheese spoilage bacteria.

This study aimed to evaluate a polyphenol extract from olive oil by-product, to improve the preservation of "Fior di latte" cheese during storage. Nine batches of "Fior di latte" cheese were manufactured in a local dairy production plant: three batches treated with 250 µg/mL of phenols from olive oil by-products in the governing liquid; three batches treated with 500 µg/mL of phenols in the governing liquid, and three untreated batches as the control group. The phenols effectively retarded the growth of Pseudomonas fluorescens and Enterobacteriaceae, acting mainly on the lag phase of the microorganisms, resulting in the prolongation of the time needed to reach the acceptable microbial limit. The delay in the growth of the spoilage bacteria was considered to indirectly influence the sensorial acceptability threshold, recorded through survival analysis. The combination of the two above-mentioned factors promotes the extension of "Fior di latte" cheese shelf life. by >2 and 4 days, for PA and PB respectively.

Olive mill wastewater phenolic concentrate as natural antioxidant against lipid-protein oxidative deterioration in chicken meat during storage.

Considering that many plant-derived substances show antioxidant and antimicrobial properties, natural antioxidant administered through feed in livestock animals could increase the shelf life of meat and meat products. The aim of this work was to study the effect of olive oil by-products on chicken meat lipid and protein oxidation and oxidative stability during storage. Two hundred and ninety-seven 22-day-old fast growing (Ross 308) female chicks were randomly assigned to three experimental grower-finisher diets: i) a basal control diet (CTR), ii) CTR diet supplemented with a low dosage (4.8%) of olive mill wastewater extract (L-OW) and iii) CTR diet supplemented with a high dosage (9.9%) of olive mill wastewater extract (H-OW). Breast meat of animals belonging to each experimental group was sliced, overwrapped with oxygen-permeable packaging and analysed at three different storage times (zero, three and seven days). At the three sampling times considered, for all samples, colour coordinates (a*), saturation index, Hue angle, peroxide value, thiobarbituric reactive substance, carbonyl assay and the oxygen radical absorbance capacity determinations were performed. No differences in colour were detected among the groups in all the sampling times considered. In conclusion, the supplementation of chicken diet with olive mill wastewater extract (OW) affected oxidation of meat, retarding lipid and protein oxidation and improving antioxidant activity during storage.

Dietary supplementation with olive mill wastewaters induces modifications on chicken jejunum epithelial cell transcriptome and modulates jejunum morphology.

BACKGROUND: The Mediterranean diet is considered one of the healthier food habits and olive oil is one of its key components. Olive oil polyphenols are known to induce beneficial effects in several pathological conditions, such as inflammatory bowel disease, and to contrast the proliferation of cancer cells or hypercholesterolemia. Polyphenols are also present in waste products derived from the olive industry: olive mill wastewaters (OMWW) are rich in polyphenols and there is an increasing interest in using OMWW in animal nutrition. OMWW are attributed with positive effects in promoting chicken performance and the quality of food-derived products. However, a tissue-specific transcriptome target analysis of chickens fed with OMWW has never been attempted. RESULTS: We explored the effect of dietary OMWW on the intestinal function in broilers. A morphological analysis of the jejunum revealed that OMWW reduced crypt depth, whereas no significant modifications were observed for villus height and the villus height/crypt depth ratio. An RNA Sequencing analysis was performed on isolated, intestinal, epithelial cells and 280 differentially expressed genes were found using a count-based approach. An enrichment analysis revealed that the majority of up regulated genes in the OMWW group were over-represented by the regulation of viral genome replication-related GO-Terms, whereas down regulated genes were mainly involved in cholesterol and lipid metabolism. CONCLUSIONS: Our study showed how an industrial waste product can be recycled as a feed additive with a positive relapse. OMWW dietary supplementation can be a nutritional strategy to improve chicken performance and health, prevent intestinal damage, enhance innate immunity and regulate cholesterol metabolism and fat deposition.

Thermal Inactivation and Growth Potential of Listeria Monocytogenes in Smoked Tench.

An experimental study for the evaluation of Listeria monocytogenes inactivation during a hot smoking process in tench was performed using Listeria innocua strains. Furthermore, the survival of L. monocytogenes in smoked tench was determined after post-processing in contaminated samples, evaluating the growth potential during storage. L innocua was not detected after the smoking process. In the challenge test, the growth potential of L. monocytogenes was 5.68 log colony forming unit g-1. The results showed that hot smoking at an inner temperature around 72°C is able to eliminate the microorganism. Nevertheless, the product is able to support the growth of the pathogen if post-process contamination occurs, as the food is suitable for Listeria multiplication. Product recontamination should be prevented by means of appropriate application of hygiene measures.

Antimicrobial Activity of Olive Mill Wastewater Extract Against Pseudomonas Fluorescens Isolated from Mozzarella Cheese.

Olive mill wastewater polyphenol extract was tested for antimicrobial activity against 64 strains of Pseudomonas fluorescens responsible for mozzarella discolouration. The extract showed a minimum inhibitory concentration (MIC)50 value of 5 mg/mL and a MIC90 value of 7 mg/mL. The MBC50 and MBC90 values corresponded to 6 and 8 mg/mL, respectively. The MIC concentration (7 mg/mL) was demonstrated to have a bacteriostatic effect while maintaining the bacterial concentration on the levels of the inoculum for 48 hours. The 3/2 MIC concentration was responsible for four logs CFU/mL depletion in colony count after 24 h. As the extract concentration decreased from MIC value, no inhibitory effects were recorded.