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The ginger oil traded worldwide could come from various sources. Standard quality is the most critical aspect of ensuring customer safety. This study aims to develop an analytical method for red ginger oil (RGO) authentication. Chemical compositions of red ginger oil were determined by Gas Chromatography-Mass Spectrometry (GC-MS). The Fourier Transform Infrared Spectroscopy (FTIR) coupled with multivariate analysis (discriminant analysis (DA), partial least square (PLS), and principal component regression (PCR) were used to identify and quantify the adulterant. The total terpenoid compounds were 55.72%, with the percentage of monoterpenes at 34.29% and sesquiterpenes at 21.43%. E-Citral (19.01%), Z-Citral (14.82%), Geranyl Acetate (11.90%), Geraniol (9.56%), 1,8-Cineole (5.84%), and camphene (4.92%) were identified as the main constituents. The best PLS model for quantifying the level of palm oil in RGO was at the wavenumber 3100–2700 cm–1, while the region of 3100 – 2700 and 1850 – 650 cm–1 was suitable for detection of soybean adulterants. FTIR spectroscopy coupled with chemometrics produced accurate and fast authentication of red ginger oil without the used solvent. Then, the GC-MS technique could identify the chemical constituents present in the red ginger oil.

Mardina V, Harmawan T, Fitriani, Sufriadi E, Febriani, Yusof F. 2020. Euthynnus affinis viscera-an alternative source for protease and lipase enzymes: Characteristic and potential application as destainer agent. Biodiversitas 21: 5858-5864. This study described the characterization of protease and lipase, extracted from the viscera of Euthynnus affinis and their applications as stains removal. Enzyme increased a few folds (3.99 folds for protease and 2.45 folds for lipase) after the submission to partial purification processes by ammonium sulfate precipitation and dialysis. Molecular mass assessment by SDS-PAGE showed that the enzyme solution contains three protein bands, estimated at 70, 42, and 24 kDa for protease and 32 kDa for lipase. Zymography confirmed the presence of protease and lipase in the sample. The protease and lipase exhibited optimum activities at pH 7.0 and pH 8.0 respectively, active at temperatures from 35 to 75oC with optimal activities at 65oC for both. The enzymes were stable at alkaline pH after 90 minutes (mins) of incubation and were also stable up to 65oC, showing the remaining activities of more than 70%. Samples containing protease and lipase as de-staining agents were tested by their ability to remove blood and palm oil stains, with results proving they were effective. Overall, the study showed that crude fish visceral extract contains protease and lipase and is useful in removing household stains, and could substitute for commercial detergents.

Objective The activity of enzymes participating in the systems of antioxidant protection was assayed in the peel and pulp of sunflower. The essential roles of proteases in food stimulate research to find other sources of the enzyme especially from non-conventional sources. In the present work, we study several biochemical parameters in the pulp and peel of sunflower. Methods Pulp and peel of sunflower was extracted, antioxidant enzymes and non-enzymatic antioxidant were measured. Alkaline protease was measured and purified from pulp in sunflower. Results High carbohydrate concentration, beta-carotene, catalase and ascorbate peroxidase activities, free radical scavenging capacity and free flavonoid content were observed in the peel of sunflower. Whereas, MDA and ceruloplasmin activities were high in the pulp of sunflower. Conclusions The present study concluded that peel in sunflower are strong radical scavengers and can be considered as good sources of natural antioxidants for medicinal and commercial uses. Further analysis showed that protease activity was a significantly high in the pulp compared to the peel.

Background The genetic features indicate a crucial role in nephrolithiasis. The present study was aimed to investigate the role of Glutathione-S-transferase Mu (GSTM1), Glutathione-S- transferase Theta (GSTT1) and endothelial nitric oxide synthase (eNOs) gene polymorphism in nephrolithiasis. Methods We involved a case-control study in which 480 individuals were divided into 240 healthy control and 240 patients with nephrolithiasis. For each patient and control, we measured biochemical criteria, levels of glutathione S-transferase, eNOs, GSTM1, GSTT1genes and eNOS genes polymorphism by PCR-RFLP. Results GSTM1 and GSTT1 null genotypes are not a risk features for nephrolithiasis. The eNOS frequency GG, GT, and TT genotypes by using Ban II enzyme as restriction enzyme were found to be (48.33, 36.67, and 15.00) %. The eNOS frequency TT, GT, and GG genotypes by using the Ban II enzyme as restriction enzyme were found to be 15.84, 25.83, and 58.33%, respectively. The result showed an increase in serum eNOs levels were in the patient's group comparing to control. Conclusions This work is the first in the literature to study the relation between eNOs genes polymorphisms and nephrolithiasis. The results conclude that TT genotypes in the eNOs genes are associated with an increase the oxidative stress in patients.

Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.

This study focused on the production of a novel multi-CLEA comprising the enzyme activities of lipase and protease from fish viscera. A multi-CLEA is a single biocatalyst that can catalyze separate unrelated reactions, but these reactions can be conducted in one application. Tests pertaining to the effect of various additives on the multi-CLEA’s activity were performed. Response Surface Methodology’s Face Centered Central Composite Design (FCCCD) was employed to optimize the preparation parameters of the multi-CLEA in an aqueous medium. It was found that 55% (w/v) of ammonium sulfate, 65 mM of glutaraldehyde, and 0.113 mM of bovine serum albumin were the optimum levels of additives to prepare the multi-CLEA with the protease and lipase recovery activity of 43.82% and 99.91%, respectively. Multi-CLEAs were found to retain an average of more than 34% of the initial activity after five consecutive batches for both enzymes. Finally, the multi-CLEA was utilized to catalyze two reactions: improved washing process and biodiesel production. The stain removal percentage of a commercial detergent was improved by 67.78% after adding multi-CLEA. In addition, the multi-CLEA catalyzed biodiesel production from vegetable oil with a percentage conversion of 51.7%. Such results demonstrated that the multi-CLEA is a promising catalyst for biotechnological applications.

Background: Acute leukaemia is characterized by fast growth of abnormal clones of haemopoietic precursor cells inside bone marrow leading to undue accumulation in the bone marrow. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Materials and Methods: The study concerned 50 children diagnosed with ALL (mean age, 8.55±2.54) compared to 40 healthy controls (mean age, 8.00±1.85). The Hb, serum copper , ceruloplasmin oxidase, advanced oxidation protein products (AOPPs), total antioxidant activity (TAA) and protein were measured in all groups.One proteinous component was isolated by gel filtration chromatography from the precipitate produced by polyethylene glycol. Results: Significantly higher levels of AOPP, copper and decrease in total antioxidant activity were noted in the cases. Statistical analysis also showed a significant increase (p

Background It is assumed that genetic factors play crucial role in nephrolithiasis. The present study was conducted to explore the role of Human Transcription Factor-7 like-2 (TCF7L2) β-defensin (DEFB1) and CD14 gene polymorphism in development and progression of nephrolithiasis. Methods The genotypes of TCF7L2, DEFB1 and CD14 polymorphism were determined in 240 nephrolithiasis patients and 240 healthy controls by restriction digestion method of PCR. The levels of serum TCF7L2, DEFB1, CD14, uric acid and other biochemical parameters were measured both in nephrolithiasis patients and healthy control. Results The patients and control groups showed 30% and 50% 1654 AA DEFB1 genotype respectively. The Allele frequency in case of patient's group was 63.67% while in control group it was 36.33%. The mean serum DEFB1 levels of the patients and control groups attained were 115.66 and 239.43 pg/mL respectively. The allele frequency of TCF7L2 in patients and controls were 44.17% and 70.0% for C-allele, 55.83% and 30.00% for T-allele respectively. The mean of serum TCF7L2 levels were significantly decreased in patients compared to control group. Conclusions The present findings are first of its class that validates a considerable connection of DEFB1 and TCF7L2 gene polymorphisms with nephrolithiasis and could probably act as indicators to estimate the risk associated to nephrolithiasis.

Background The intron 5 insertion/deletion polymorphism of Alpha-2-macroglobulin receptor-associated protein gene (Alpha-2-MRAP) has been implicated in numerous diseases. The current study was designed to analyze the association of intron 5 insertion/deletion polymorphism of Alpha-2-MRAP with nephrolithiasis patients. Methods PCR was conducted on genomic DNA of patients and control to look for Alpha-2-MRAP insertion/deletion polymorphism. Besides that, serum level of Alpha-2-MRAP, oxidative stress marker myeloperoxidase, Malondialdehyde (MDA), Advanced oxidation protein products (AOPP), and uric acid were determined. Results The D and I allele frequencies were 57.50% and 42.50% in patients, 77.50% and 22.50% in control, individually. The result showed that II genotype was associated with nephrolithiasis patients group. A significant decrease was observed in serum Alpha-2-MRAP,myeloperoxidase and TAS,while TOS,OSI,MDA,AOPP and uric acid were substantially increased in II and ID when compared to DD genotype in patients with nephrolithiasis. Conclusion Our results demonstrate for the first time that patients with II genotype had an increased risk of stones. Also, the results demonstrate that I allele of the 5 insertion/deletion polymorphism in the Alpha-2-MRAP gene is related with an increase of oxidative stress in nephrolithiasis patients and may possibly impose a risk for cardiovascular diseases in patients with II genotype of Alpha-2-MRAP.

Objective To prove probable relations between serum E3 SUMO-protein ligase NSE2 (NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients. Methods A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA. Results A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased. Conclusions The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

Commercially available bromelain is prepared by performing a tedious and costly purification method that yields bromelain at different degrees of purity. In the current study, a gene encoding stem bromelain from Ananas comosus was amplified using polymerase chain reaction. This bromelain gene was initially cloned into the pENTR/TEV/D-TOPO vector before being sub-cloned into the pDEST17 expression vector. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the BL21-AI Escherichia coli strain. The recombinant bromelain was then purified in a single step using immobilized metal affinity chromatography, specifically a Ni-NTA spin column. The purified recombinant bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant bromelain exhibited optimum activity at pH 4.6 and 45 °C.

Euthynnusaffinis is one of tuna species that distributed throughout the world. Their visceral accounts for approximate less than 20% of body weight and its value are still underutilized. This study screened four the potential individual visceral organ to produce protease and lipase enzyme viz. spleen, stomach, pancreas, and liver. Among the individual visceral organ, liver exhibited the highest source for protease activity (57.786 U/ml), while pancreas revealed as the best source for lipase activity (69.425 U/ml). Therefore, the finding of alternative sources for protease and lipase in this study might converted visceral waste by product to generate protease and lipase enzyme that may be applied in biotechnological process.

Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase, glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries. The extraction of high active lipase from CPH has been done under optimum conditions. The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvents.

Halal slaughtered chicken is a major concern among Muslims. Currently, stunning methods has been adopted worldwide aiming at maximizing production. However, stunning treatment which is not in compliance with the Islamic teaching will end up with unlawful chickens for Muslim consumption. In this study, proteomics methods have been used to identify potential biomarkers for the effects of electrical stimulation on the protein profiles of chicken’s brain. The protein profiles were constructed in order to detect any differences in the protein expression and modifications. The electrical stimulation was conducted with different current and voltage. The different voltages studied were 10, 40, and 70 V while the values for current studied were 0.25, 0.5, and 0.75 A. The profiles from these electrically stimulated chicken were compared to the 0 A, 0 V non-stunned sample. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF) identification, aldolase C, actin-related protein 2 homolog (ARP2), and enolase 1 were identified to be positively present in the brain sample of electrically stimulated samples. The over expression of the proteins were further confirmed at the transcriptional level by real-time PCR. Results from MALDI-TOF and real-time PCR were in agreement. Therefore, this identification of biomarkers for electrically stimulated chicken’s brain provides a novel approach on differentiating the stunned chickens from the non-stunned ones.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis aliene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K. value of 4.02 ± 0.64 ttM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress. © 2007 Elsevier Masson SAS. All rights reserved.

Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.

As the living cytoplasm of laticiferous cells, Hevea brasiliensis latex is a rich blend of organic substances that include a mélange of proteins. A small number of these proteins have given rise to the problem of latex allergy. The salient characteristics of H. brasiliensis latex allergens that are recognized by the International Union of Immunological Societies (IUIS) are reviewed. These are the proteins associated with the rubber particles, the cytosolic C-serum proteins and the B-serum proteins that originate mainly from the lutoids. Procedures for the isolation and purification of latex allergens are discussed, from latex collection in the field to various preparative approaches adopted in the laboratory. As interest in recombinant latex allergens increases, there is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy.

The Lowry protein assay is widely used to quantify proteins in the laboratory. As the assay is susceptible various interfering substances, the protein test sample is frequently first purified by precipitating the proteins using trichloroacetic acid (TCA). However, TCA precipitates proteins poorly in the presence of sodium dodecyl sulfate (SDS), a detergent very commonly used in biological research. The inhibitory effect of up to 1% SDS on protein precipitation is effectively overcome by a combination of 5% TCA and 1% phosphotungstic acid (PTA). The TCA/PTA mixture also precipitates proteins readily in the presence of other detergents such as 0.1% Tween 20, Triton X-100 or Nonidet P-40. TCA/PTA has the capacity to precipitate a wider cross-section of proteins than TCA alone, the combination of acids remaining effective in the presence of SDS and other detergents.

IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39−42 % identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.

Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 microgram ml-1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 micrograms) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 micrograms ml-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 microgram ml-1 protein present in 6 ml of solution can be assayed.

Methods In this study, proteomics methods have been used to study the effects of different currents and voltages used to stun chickens. Protein profiles of chicken hearts were constructed to detect differences in protein expression and modification. The different voltages studied were 10 V, 40 V and 70 V, while the currents examined were 0.25 A, 0.5 A, and 0.75 A. The profiles obtained from these stunning conditions were compared to the non-stunned (0 A, 0 V) sample. Results Proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF) identified troponin I and alpha cardiac muscle actin 1 in the electrically stimulated heart samples. The overexpression of the proteins was further confirmed at the transcriptional level by Real Time PCR. Conclusion The results from MALDI-TOF and Real Time PCR agreed; therefore, this method for identifying biomarkers of electrically stimulated chicken hearts provides a novel approach for differentiation the hearts of increased electrically stimulated chickens from those of non-stunned chickens.

Rationale Identification of Hev b 7, a 43 kDa latex allergen homologue of patatin, did not adequately explain the frequent observations of IgE antibody reactivity to a 42–46 kDa protein in serum from latex-allergic patients. We thus hypothesized the existence of another allergenic Hevea protein of a similar molecular weight. We report on the isolation and purification of a 42.98 kDa latex glycoprotein showing homology to Early Nodule Specific Protein in legumes. Methods Hev b 13 was isolated from Hevea brasiliensis latex B-serum by gel filtration and ionic exchange chromatography. Binding of human IgE to Hev b 13 and inhibition of IgE binding by patatin were studied by Western immuno-blots analysis. The cDNA encoding Hev b 13 was isolated by RT-PCR and cloned. Results The protein predicted from the Hev b 13 cDNA sequence has 391 amino acids. The mature protein possessed a deduced molecular weight of 40.40 kDa and pI of 5.0. Prevalence of IgE antibody reactivity to native Hev b 13 was 78% (51/65) in latex-sensitized healthcare workers and 77% (10/13) in latex-sensitized spina bifida children. IgE-binding to Hev b 13 was inhibited by the potato tuber patatin but lost when the carbohydrate moiety of patatin was removed. This suggested that the IgE-specific epitope might reside on the glycan. Conclusions The lipolytic esterase, Hev b 13, is a major allergen of Hevea brasiliensis latex. The principal IgE-specific epitope of the glycoprotein appears to be located on its carbohydrate moiety.