Betel (Piper betle L.) Leaf Ethanolic Extract Dechlorophyllized by Sedimentation: Uses in Conjunction with Cold Plasma and for Preparation of Active Packaging to Extend the Shelf-Life of Nile Tilapia Slices

Abstract

Sedimentation process was used to remove chlorophyll from betel and chaphlu leaf ethanolic extracts (BLEE and CLEE, respectively) by mixing the extract with water. The process showed a remarkable reduction in chlorophyll content for both extracts. No differences in chlorophyll contents, total phenolic content (TPC), or antioxidant activities were observed between the dechlorophyllized fractions using different extract/water ratios (p > 0.05). Liquid Chromatography-Mass Spectrometry (LC/MS) profiling showed that BLEE dechlorophyllized using the extract/water ratio of 1:1 (BLEE-DC1) had higher phenolic compounds than CLEE dechlorophyllized counterpart (CLEE-DC1). Isovitexin was the most abundant compound identified in the BLEE-DC1, while vitexin 4′-O-galactoside was the most prevalent in CLEE-DC1. BLEE-DC1 was generally more heat stable than CLEE-DC1. Different solvents were used for the dechlorophyllization of BLEE in comparison with sedimentation process. Sedimentation reduced the chlorophyll content and color of BLEE more efficiently (p < 0.05), while antioxidant and antibacterial activities were enhanced (p < 0.05). BLEE dechlorophyllized by the sedimentation (BLEE-SED) had lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as compared to other dechlorophyllized extracts. Lower microbiological and chemical changes took place in Nile tilapia slices treated with BLEE-SED at 400 and 600 ppm after 12 days of storage at 4˚C. BLEE-SED was loaded in liposomes at 1 and 2% (w/v) using two different methods, namely thin film hydration (TF) and ethanol injection (EI) methods.Liposomes loaded with 1% BLEE prepared by TF method (L/BLEE-T1) had the smallest particle size, paler color and was more stable than the other prepared liposomes ascertained by its lowest zeta potential and polydispersity index (p < 0.05). The highest encapsulation efficiency (EE) and lowest releasing efficiency (RE) were also found with L/BLEE-T1. L/BLEE-T1 showed higher antioxidant stability than unencapsulated BLEE prepared at equivalent amount based on EE (U/BLEE-T1) after in vitro gastrointestinal tract digestion. L/BLEE-T1 could be therefore an efficient delivery system for improving stability of antioxidant activities of BLEE. L/BLEE-T1 showed enhanced antibacterial activity as witnessed by lower MIC and MBC (p < 0.05). L/BLEE-T1 also caused larger inhibition zones, lower triphenyl-2H tetrazolium chloride (TTC) dehydrogenase activity, and higher release rates of K+ and Mg2+ ions to the tested bacteria (p < 0.05). Additionally, scanning electron microscopic (SEM) images showed deformations and perforation on cell walls of the tested bacteria after treatment with L/BLEE-T1. At 400 ppm, L/BLEE-T1 combined with modified atmospheric packaging (MAP) (CO2: Ar: O2 = 60: 30: 10) and non-thermal plasma (NTP) (80 KV-RMS for 5 min) exhibited the highest inhibition effect (p < 0.05) toward the challenged bacteria over 15 days of storage at 4˚C. Nile tilapia slices treated with L/BLEE-T1 or U/BLEE-T1 at 400 ppm, packed under MAP (CO2: Ar: O2 = 60:30:10) and subjected to NTP for 5 min (L/BLEE 400/MAP-NTP and U/BLEE-400/MAP-NTP, respectively) had the lowest microbial and chemical changes during storage of 12 days at 4˚C (p < 0.05). Lipid oxidation was lower in these samples, as indicated by more retained polyunsaturated fatty acids and lower lipid oxidation based on Fourier transform infrared (FT-IR) spectra. Overall likeness scores were similar (p > 0.05) between all the samples at day 0 of storage. However, only L/BLEE-400/MAP-NTP and U/BLEE-400/MAP-NTP were still sensorially acceptable after 12 days of storage. Incorporation of dechlorophyllized BLEE at 1 and 2% enhanced elasticity and heat-seal ability of the gelatin/chitosan blend films, particularly, those plasticized with glycerol (GLY). Ultraviolet and visible light barrier abilities along with water vapor permeability were improved (p < 0.05) for films containing 2% BLEE. Swelling and water solubility of the films were lessened with augmenting BLEEconcentrations (p<0.05). Antioxidant and antibacterial activities were surged as BLEE levels incorporated increased. Type of plasticizer had no effect on antioxidant and antibacterial activities of films (p > 0.05). SEM images showed smooth homogenous surface and cross-section in film sample devoid of BLEE, while those incorporated with BLEE had slightly rough surface and cross-section, but the roughest surface and cross section were found for films incorporated with L/BLEE. FT-IR spectra revealed the difference in peaks and patterns between films without and with BLEE. Addition of BLEE lowered thermal stability of film associated with looser structure of film. Pouches made from gelatin/chitosan blend plasticized with GLY and incorporated with BLEE at 2% (B-GLY) were used to pack shrimp oil. They could prevent lipid oxidation more efficiently than lower density polyethylene (LDPE) pouches as ascertained by less peroxide values, thiobarbituric acid reactive substances, and astaxanthin decomposition. B-GLY could be thus used as active packaging to retard lipid oxidation in shrimp oil. Gelatin/chitosan solutions incorporated with BLEE at varying concentrations were electro-spun on polylactic acid (PLA) films. Gelatin/chitosan nanofibers (GC/NF) with different morphologies, as indicated by SEM, were formed. PLA films coated with GC/NF added with BLEE showed antioxidant and antibacterial activities. Lowered water vapor permeability and enhanced mechanical properties were achieved for GC/NF coated PLA films (p<0.05). Microbial growth and lipid oxidation of Nile tilapia slices packaged in PLA films coated with GC/NF containing 2% BLEE were more retarded than those packaged in low density polyethylene (LDPE) bags over the refrigerated storage of 12 days. Based on microbial limit, the shelf-life was escalated to 9 days, while the control had shelf-life of 3 days. Therefore, such a novel film/bag could be a promising active packaging for perishable fish and fish products.