Jelly Fig Research Articles

Thermostable ficin from jelly fig (Ficus pumila var. awkeotsang) latex: purification, identification and characterization

The achenes/seeds of endemic jelly fig (Ficus pumila var. awkeotsang) fruit have been applied to prepare a traditional beverage in Taiwan. Upon fruit harvest, jelly fig latex exuded from stalks was discarded. Protease activity was monitored in its latex. Proteases capable of hydrolyzing proteins have many application aspects based on diverse characteristics. Commercial plant proteases are frequently from latex. The latex protease of jelly fig, termed FaFicin, was purified to homogeneity with a molecular mass of ~32 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to liquid chromatographic-tandem mass spectrometric analyses, the expected protein band of protease was matched to ficin A, ficin B or chymopapain from common fig or papaya. Iodoacetamide, an inhibitor of cysteine protease, inhibited its protease activity completely. Hence FaFicin was identified as a papain-like cysteine protease (PLCP), exhibiting more than 80% and 70% activity as assayed at pH5-8 and 40-70 °C, respectively. It maintained ~89% of initial activity after 120 min at 55 °C and pH7. Moreover, FaFicin could degrade the myosin and actin of meat, and clot milk. The ficin FaFicin was obtained, purified and identified as a PLCP member from agricultural waste: jelly fig latex. It possessed activity under a wide range of pH values and temperature, and exhibited excellent thermostability. Based on its initial evaluation as a meat tenderizer and milk clotting reagent, the application of FaFicin was possible, which may extend utilization of jelly fig. © 2022 Society of Chemical Industry.

Fast Dissolving Electrospun Nanofibers Fabricated from Jelly Fig Polysaccharide/Pullulan for Drug Delivery Applications.

The fast-dissolving drug delivery systems (FDDDSs) are developed as nanofibers using food-grade water-soluble hydrophilic biopolymers that can disintegrate fast in the oral cavity and deliver drugs. Jelly fig polysaccharide (JFP) and pullulan were blended to prepare fast-dissolving nanofiber by electrospinning. The continuous and uniform nanofibers were produced from the solution of 1% (w/w) JFP, 12% (w/w) pullulan, and 1 wt% Triton X-305. The SEM images confirmed that the prepared nanofibers exhibited uniform morphology with an average diameter of 144 ± 19 nm. The inclusion of JFP in pullulan was confirmed by TGA and FTIR studies. XRD analysis revealed that the increased crystallinity of JFP/pullulan nanofiber was observed due to the formation of intermolecular hydrogen bonds. The tensile strength and water vapor permeability of the JFP/pullulan nanofiber membrane were also enhanced considerably compared to pullulan nanofiber. The JFP/pullulan nanofibers loaded with hydrophobic model drugs like ampicillin and dexamethasone were rapidly dissolved in water within 60 s and release the encapsulants dispersive into the surrounding. The antibacterial activity, fast disintegration properties of the JFP/pullulan nanofiber were also confirmed by the zone of inhibition and UV spectrum studies. Hence, JFP/pullulan nanofibers could be a promising carrier to encapsulate hydrophobic drugs for fast-dissolving/disintegrating delivery applications.

Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase.

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans.

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50–80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 μmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21–94%). The enzyme hydrolyzed 70–84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Electrophoretic Ink Display Prepared by Jelly Fig Pectin/Gelatin Microspheres

A brand new Bio-Electronic ink (Bio-E ink) display device was prepared and characterized in this study. Semiconductor material, copper phthalocyanine (CuPc) was modified by cationic surfactants, cetylpyridinium chloride (CPC), as the core material, and the shell of capsule was prepared by jelly fig pectin, gelatin and sodium dodecyl sulphate (SDS). Here, jelly fig pectin was provided as the shell material for the first time. Chemical structure of the modified CuPc was characterized by Fourier Transform Infrared Spectrometer (FTIR). The core-shell microcapsules were achieved by coacervation method in an oil/water (O/W) emulsion system. The particle size and morphology of microcapsules were affected by the concentrations of SDS and pH values of the O/W emulsion system. A new microcapsule-based electrophoretic display device was presented. Its image display ability of the microcapsules electrophoretic device was presented as appropriated electric power was applied, and the response time was 0.06 sec under 0.1 V/mm of electric field. Moreover, we found that its image contrast ratio of display device was influenced by the particle sizes of the microcapsules.

G2/M arrest and apoptosis of human colorectal cancer cells induced by water extract from residues of jelly fig achene.

Jelly fig (Ficus awkeotsang) achenes have been utilized to prepare a traditional drink in Taiwan. Herein, we evaluated the effect of water extract from jelly fig seed residues (WERJFA) on cancer cells. WERJFA could inhibit the growth of human colorectal cancer cells, COLO205 and HT29 in both dose- and time-dependent manners. The flow cytometric analysis with propidium iodide (PI) showed that WERJFA primarily arrested COLO205 and HT29 cells at the G2/M phase of cell cycle as the concentration reached to at least 0.5 mg/ml. WERJFA induced apoptosis of these two cell lines, as evidenced by annexin V-FITC/PI and 4', 6-diamidino-2-phenylindole (DAPI) staining using flow cytometry and confocal microscopy, respectively. Reactive oxygen species (ROS) production and the loss of mitochondrial membrane potential in WERJFA-treated cells were detected by flow cytometry with H2DCF-DA and 5,5', 6,6'-Tetrachloro-1, 1', 3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). Our results showed that WERJFA exerted anti-proliferative and apoptotic effects on colorectal cancer cells. WERJFA arrested cell cycle, and caused apoptotic death in these cancer cells possibly via mitochondrial pathway involved with exceeding ROS level.

Electrophoretic Ink Display Prepared by Jelly Fig Pectin/Gelatin Microspheres

A brand new Bio-Electronic ink (Bio-E ink) display device was prepared and characterized in this study. Semiconductor material, copper phthalocyanine (CuPc) was modified by cationic surfactants, cetylpyridinium chloride (CPC), as the core material, and the shell of capsule was prepared by jelly fig pectin, gelatin and sodium dodecyl sulphate (SDS). Here, jelly fig pectin was provided as the shell material for the first time. Chemical structure of the modified CuPc was characterized by Fourier Transform Infrared Spectrometer (FTIR). The core-shell microcapsules were achieved by coacervation method in an oil/water (O/W) emulsion system. The particle size and morphology of microcapsules were affected by the concentrations of SDS and pH values of the O/W emulsion system. A new microcapsule-based electrophoretic display device was presented. Its image display ability of the microcapsules electrophoretic device was presented as appropriated electric power was applied, and the response time was 0.06 sec under 0.1 V/μm of electric field. Moreover, we found that its image contrast ratio of display device was influenced by the particle sizes of the microcapsules.

Weakness of Aqueous Polysaccharide Gels Prepared from Jelly Fig

Weakness of Aqueous Polysaccharide Gels Prepared from Jelly Fig

Factors Affecting the Gelation Behavior of Aqueous Components Extracted from Jelly Fig

Factors Affecting the Gelation Behavior of Aqueous Components Extracted from Jelly Fig

Rheological Properties of Aqueous Polysaccharide Sols and Gels Prepared from Jelly Fig

Rheological Properties of Aqueous Polysaccharide Sols and Gels Prepared from Jelly Fig

The Texture Profiles of Jelly Fig Gel

The Texture Profiles of Jelly Fig Gel

Determination of N-Glycosylation Site and Glycan Structures of Pectin Methylesterase in Jelly Fig (Ficus awkeotsang) Achenes

Pectin methylesterase (PME) in jelly fig (Ficus awkeotsang) achenes is an N-glycosylated enzyme responsible for the gelation of jelly curd. A recombinant jelly fig PME was overexpressed in Escherichia coli and confirmed by immunodetection and LC-nanoESI-MS/MS analysis. To identify the N-glycosylation site, native PME and its deglycosylated and recombinant forms, which lacked glycan, were purified and subjected to comparative MALDI-MS mapping of the corresponding tryptic fragments. The results showed that N-glycosylation occurred at Asn(153) of the mature jelly fig PME, the only glycosylation site predicted by its sequence analysis. The major N-glycans released from the native PME by PNGase F were identified by MS/MS analyses as xylosylated, noncore fucosylated pauci-mannose, and complex-type structures. Molecular modeling of the 3D structure of jelly fig PME indicated that the N-glycan was putatively attached to the back region of the active site of this enzyme.

Purification, Cloning, and Identification of Two Thaumatin-like Protein Isoforms in Jelly Fig (Ficus awkeotsang) Achenes

Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of beta-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments.

Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes.

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.

Influence of Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achenes on Pectinesterases and Cloud Loss of Fruit Juices

ABSTRACT: Citrus and jelly fig achene pectinesterase (PE) (1 U/mL) activity was remarkably inhibited by PE inhibitor (PEI) (2 mg/mL) from jelly fig (Ficus awkeotsang Makino) achenes, in the absence or presence of 0‐ to 500‐mM salts or 0 to 30% of sugars. Changes in temperature (20 to 40 °C) apparently decreased the activity of citrus PE in PE‐PEI reaction mixture. Enzyme‐activity curves for jelly fig PE alone and for that in combination with PEI were parallel. Activity of crude PEs (1 U/mL) from tomato, apple, asparagus, and guava was reduced remarkably. Cloud loss of fresh tomato juice, apple juice, and papaya juice was greatly inhibited by PEI (1 mg/mL) during 12‐wk storage.

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes.

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.

De‐esterification and Transacylation Reactions of Pectinesterase from Jelly Fig (Ficus awkeotsang Makino) Achenes

ABSTRACT: Pectinesterase (PE) was isolated from jelly fig (Ficus awkeotsang Makino) achenes, then the optimal conditions for de‐esterification and transacylation reactions were determined. Molecular weight of pectin (DE = 62.8 %) when reacted with PE at pH 6.5 and 45 °C in 0.2 M NaCl for 20 min remarkably increased from the original 72 kDa to 410 kDa, as determined by Fractogel TSK 65(S) gel permeation chromatography. Prolonging the incubation time of pectin‐PE mixtures to 2 and 4 h also increased the molecular weights of pectin. Therefore, transacylation reaction was considered to occur and to increase the molecular weight of pectins when de‐esterification reaction was catalyzed by pectinesterase (PE).

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang).

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.