Characterization Of Allergens From Dust Mite (Tyrophagus Putrescentiae)

Hypersensitivity to dust mite allergens is one of the most common allergic reactions in the world with estimated 10% of the general population and 90% of individuals suffering from allergic asthma are sensitive to dust mites. Tyrophagus putrescentiae (TP) represents one of the common storage mites which has a worldwide distribution with particularly highly prevalence in tropical and subtropical regions and its explicit allergenic importance in causing mite sensitization has been well documented. In an attempt to evaluate the allergenicity of T. putrescentiae, few immunological tests have been performed on T. putrescentiae crude extracts by using sera from allergic subjects. Dot blot screening revealed that 49.7% of 141 patient sera showed the presence of specific IgE towards TP mite components. There were at least 15 IgE binding components present in TP with molecular weights ranging from 10 to 150 kD with 15 and 77 kD appearing to be major allergens observed after immunoblotting. At the same time, the cross-reactivity studies were carried out in an effort to establish the antigenic relationship between T. putrescentiae and eight other mite species which is important for accurate allergy diagnosis as well as effective immunotherapy for allergic patients. Although most of the mites' allergens share some degree of allergenic cross-reactivity or epitopes with T putrescentiae, those mites somehow also contain unique allergens or epitopes with relatively low crossreactivity with T putrescentiae allergens. Also, cross-reactivity between T putrescentiae and other mite allergens in this study was likely to be the result from multiple sensitizations of allergic subjects to coexisting mite species particularly the principal mite species (Blomia and Dermatophagoides spp. ) in the studied environment

Bacillus Multi-strain from Malaysian Fish Sauces Demonstrating Proteolytic, Lipolytic, Esterolytic and Glutamic-acid Production Activities

Background and Objective: Aroma and taste of fish sauce are derived from complex metabolic reactions involving bacteria and enzymes that degrade proteins and lipids. A longer fermentation time is needed to fully develop the rich flavor. The objective of this study was to isolate halotolerant bacteria from Malaysian fermented fish sauce that demonstrated significant proteolytic, lipolytic, esterolytic and glutamic acid-accumulating activities. These bacteria might potentially be used as starting cultures to enhance development of flavors during fish sauce fermentation. Material and Methods: The initial screening on proteolytic activity was carried out on saline skim milk agar. Isolates with high proteolytic index and positive lipase/esterase activity were further assessed using casein-based protease activity assay. Protease and lipase activities under various conditions of strains, NaCl concentrations (20–30% w/v), temperatures (30–35 °C) and incubation times (0–120 h) were assessed and analyzed using four-way ANOVA. The p-nitrophenyl butyrate and colorimetric assays were used to determine esterase and glutamic acid accumulating activities, respectively. Results and Conclusion: Six strains isolated from budu demonstrated hydrolytic activities and identified as Bacillus spp. Proteolytic activities were averagely highest in B. licheniformis 12N3A at 25% NaCl, 30 °C and 96 h of incubation. The highest lipolytic activities were achieved with B. haikouensis 12M1F at 30% NaCl, 35 °C and 72 h of incubation. Bacillus sp. 3M3A showed the highest esterase activity of 155.48 U, while Bacillus sp. 6M2A had the highest glutamic acid accumulation at 1993.02 mol. l-1. The strains of this study have demonstrated appropriateness for use as a cocktail culture to accelerate the fermentation process and enhance the flavor profile of fish sauces. Halotolerant nature of the strains and enzymes supports their potentials for wider use in the food industry. Conflict of interest: The authors declare no conflict of interest. Introduction   Budu is a Malaysian traditional fish sauce made from anchovies fermented with salt for 6–12 m and officially recognized as a national culinary heritage. Flavor is addressed as the primary quality attribute of budu. Function prediction of budu microbial communities based on meta-genomic composition has demonstrated increased protein and lipid breakdown pathways during fermentation and these pathways are known to play critical roles in formation of flavor [1]. Through spontaneous fermentation of high-salt fish sauce, a wide range of taste and aroma-active compounds belonging to aldehydes, acids, esters, alcohols and pyrazines are gradually formed, including 2-methyl-butanal, 3-methylbutanoic acid, methyl 2-ethyldecanoate, 1-octen-3-ol and 2,6-dimethylpyrazine [1]. This is achieved through the combined activities of fish endogenous enzymes and metabolic activities of halophilic and halotolerant microbes [2]. Additionally, microbes are capable of produc-ing microbial volatile organic compounds, which may stimulate production of secondary metabolites, especially during co-cultures [3]. Fatty acids, peptides and amino acids that are released through the process of lipid and protein hydrolyses serve as precursors for aroma compounds. They can be transformed into volatile organic compounds, including aldehydes, esters, ketones, alcohols, furans, hydrocarbons, lactones, pyrazines, amines, phenols, indoles, acids and sulfur-containing compounds [4]. The medium and short-chain fatty acid esters are important volatile organic compounds that can impart pleasant fruity flavors and aromas [5] and their presence in fish sauce can cover unpleasant smells and off flavors contributed by protein-derived compounds such as dimethyl trisulfide and trimethylamines. Microorganisms involved in fish sauce fermentation may facilitate glutamic acid accumulation by converting ketoglutaric acid into L-glutamic acid under the function of glutamate synthetase or glutamic acid dehydro-genase, as well as hydrolysis of proteins into umami amino acids and peptides [6]. Figure 1 shows schematic functions of protease, lipase and esterase as well as formation of glutamic acid in fermentation of fish sauces. At the onset of fermentation, fermentation-assisting bacteria are not the prevailing microorganisms. They need longer times to adapt to high salinity, establish dominance in the microbial community and produce flavor compounds [7].  Consequently, a prolonged fermentation time is needed to produce rich, flavorful fish sauces. The extended fermentation time can lead to an increase in overall produc-tion time, which may not be desirable for the producers who try to meet market demand and generate revenue quickly. In response to this, Malaysian fish-sauce producers terminate their fermentation process after only three months even though the flavor has not fully developed. Accelerating the fermentation process is therefore important for the production efficiency and profitability of fish sauce industries. Use of starting cultures has been suggested as an effective method to enhance proteolysis during fish-sauce fermentation [8], while improving and maintaining the consistent quality of the fermented fish [9]. Addition of Marinococcus halotolerans SPQ isolated from salt crystals showed increased contents of aspartic and glutamic acids with several aroma compounds belonged to alcohol group [10]. Use of protease-producing Tetragenococcus halophi-lus has decreased fermentation time to six months and improved quantity of desirable amino acids [11]. When selecting starter cultures for fish-sauce fermentation, ease of cultivation is primarily important. It is essential for the strains to possess pro-technological metabolic character-istics such as the ability to grow and produce high-activity hydrolases that are active at high salt concentrations (20–30% NaCl). Therefore, a candidate derived from fish sauce is an excellent choice due to its adaptability, resistance to osmotic stress and ability to thrive in high salt environments. This enables them to grow more efficiently and fulfil their roles within the microbial community. Fermentation of fish, which naturally contains high protein and low sugar contents, differs significantly from conventional carbohydrate fermentation. Rather than targeting sugars, this process focuses majorly on fish protein and lipid components [12]. Therefore, proteolytic and lipolytic activities are two key characteristics that should be addressed when selecting starter candidates for fish fermentation. Release of proteases and lipases by halophilic Staphylococcus simulans PMRS35 and Haloarcula sp. isolated from salted and fermented fish [13,14] as well as M. halotolerans SPQ and Chromohalobacter sp. from salt environments [10,15] has been documented in several investigations. It is suggested that fish sauce contains a diverse enzymes-producing microbiota that contribute to the formation of taste-active compounds, making it an ideal source for the selection of starter cultures. Nevertheless, a complete assessment of various strains from budu has not been carried out, specifically for assessing production of multiple enzymes that are likely to improve budu flavor. The aim of the current study was to isolate, identify and assess bacterial cultures from budu, displaying proteolytic, lipolytic, esterolytic and glutamic-acid accumulating activities. This study provides a novel contribution to the existing literature as it assessed the bacterial enzymes involved in the flavor development of Malaysian fish sauce, an area with limited attentions. Halotolerant strains thoroughly assessed for their major abilities to degrade fish proteins and lipids make promises for potential uses in the food industry. They can provide a viable solution for improving production and fermentation of high-salt fermented foods, particularly fish sauces. Materials and Methods 2.1. Isolation of halotolerant proteolytic bacteria Fish-sauce samples from 3, 6 and 12-m fermentation tanks were used for bacterial isolation. A modified isolation approach [8] was used to concurrently identify halotolerant proteolytic bacteria during first screening. To increase the likelihood of collecting indigenous proteolytic fish-sauce origin strains, media types and salt concentrations were modified. Twenty-five grams of fish sauce were diluted with 225 ml of sterile 0.85% (w/v) NaCl and stirred for 1 min. To recover further halophilic species, three decimal dilutions of the samples were plated on four media of marine agar (MA) with 20% NaCl for bacteria tolerant to 20% NaCl; De Man, Rogosa and Sharpe agar (MRSA) with 20% NaCl for 20% NaCl-tolerant lactic acid bacteria (LAB); mannitol salt agar (MSA) with 10% NaCl for salt-tolerant Staphylococci; and nutrient agar (NA) with 30% NaCl for bacteria tolerant to 30% NaCl. Each medium received 1% of skim milk for proteolytic activity screening. The agar medium was incubated at 30 °C for 3–14 d until colonies formed. Four-quadrant streaking pattern was used to pick and streak clear zone-forming bacterial colonies with diverse morphologies onto newly prepared agar plates for purification. 2.2. Screening of protease and lipase/esterase activities using agar plate assay Prior to the screening, one loop of every single isolate was transferred to marine broth (MB) containing 20% NaCl and incubated at 30 °C for 3 d, except for colonies recovered from MSA that were grown in nutrient broth supplemented with 10% NaCl. Cells were collected by centrifugation at 4000 rpm for 10 min at 4 °C following a 3-d incubation time. To prepare pellets for resuspension, pellets were first rinsed twice with 10 ml of pH-7.4 phosphate buffered saline (PBS). For each strain, absorbance of the suspended cells at 600 nm was adjusted to 0.85 before well-diffusion method was used. For the screening of bacteria with proteolytic activity, aliquots of the bacterial suspension (50 µl) were added to a 6-mm well on agar media consisted of NA supplemented with 10% NaCl and 1% skim milk for MSA-derived strains and MA supplemented with 1% skim milk for the rest of strains. Proteolytic indices were assessed after incubating plates at 30 °C for 2 d [9]. Agar spot techniques in three various media of phenol red, tributyrin and Tween 80 agar were used to screen bacteria that produced lipase or esterase. Each medium was prepared according to [16]. Five microliters of resuspended cells were dropped onto the surface of the phenol red, tributyrin and Tween 80 agar and allowed to absorb. Inoculated plates were incubated at 30 °C for 3–14 d until visible signs of lipolysis activity were observed. The color shift of phenol-red agar from red to orange indicated lipase activity, which was caused by a slight pH drop induced by fatty acid releases. A clear zone on tributyrin agar indicated the presence of extracellular esterase. The FFAs were precipitated by calcium (giving a white zone) around colonies showing lipase and esterase activity on Tween 80 agar. Isolates demonstrating a high proteolytic index and/or indicating lipolytic activity were selected for further growth and protease activity assessment using liquid assay. 2.3. Growth assessment of the strains One loop of each selected strain was transferred to MB media and incubated at 30 °C for 48 h. After incubation, cells were centrifuged at 4000 rpm for 10 min at 4 °C. Collected pellets were washed and resuspended in PBS with an OD600 adjusted to 1.1-1.2. A 1-ml volume of the cell suspension was transferred to 40 ml of MB using 125-ml amber bottles. The mixture was incubated at 30 °C with 120-rpm agitation speed. Growth assessment was carried out by measuring aliquots of the growth media at 600 nm using Biomate 3 UV-vis spectrophotometer (Thermo-Fisher Scientific, Massachusetts, USA) at specified intervals for 216 h. Sterile MB medium was used as blank for each reading. 2.4. Extraction of the bacterial crude enzymes Cell-free supernatant (CFS) was prepared for the enzyme assays by centrifuging aliquots of the harvested growth media at 11,000 × g for 10 min at 4 °C. 2.5. Assessment of protease activity Protease activity of the isolates was determined based on the Cupp-Enyard and Aldrich method [17], where casein was used as substrate. Casein was diluted to 6.5 mg.ml-1 in 50 mM potassium phosphate buffer (pH 7.5). Temperature of the solution gradually increased to 80–85 °C with gentle stirring for nearly 10 min until a homogeneous dispersion was achieved. Casein solution was set at 37 °C prior to use. Moreover, CFS was added to 5 ml of casein solution and incubated at 37 °C for 10 min. Every tube was then treated with 5-ml solution of 110 mM trichloroacetic acid (TCA) following incubation to terminate the reaction. To verify that each tube had an identical end volume and account for the enzyme absorbance value, an equal quantity of the enzyme solution was added to the blank. Solutions were incubated at 37 °C for 30 min and then centrifuged at 4000 rpm for 5 min to remove insoluble substances. For colori-metric determination of tyrosine, 400 µl of the supernatant were immediately added with 1 ml of sodium carbonate and 0.2 ml of Folin reagent. The mixture was mixed gently and permitted to stabilize at 37 °C for 30 min. Absorbance measurement was carried out at 660 nm using spectrophoto-meter. Tyrosine concentration was determined using standard tyrosine curve. Bacterial protease unit (U) was defined as the quantity of enzyme that produced the equivalent of 1 μmol. L-1 of L-tyrosine per min in pH 7.5 and 37 °C. Isolates with significant protease activity were subjected to further analyses. The protease activity was determined using the Eq. 1   Eq.1 2.6. Effects of time, temperature and NaCl concentration on protease and lipase activities Using MB media, the selected strains were assessed for their protease and lipase activities under various temperatures (30 and 35 °C) and NaCl concentrations (20, 25 and 30%). In this study, a temperature range of 30–35 °C was selected as it represented the average temperature used in the industry. Salt concentration varied 20–30% during budu 12-m fermentation; therefore, this salt range was used to assess its effects on the strains’ enzyme activity. Protease assay was carried out using the method described in Section 2.5. To assess lipolytic activity, titration method [18] was used with emulsion of fish oil and polyvinyl alcohol at a ratio of 1:3 as the substrate for the assay. Briefly, 2.5 ml of phosphate buffer (pH 4.8) were added to 2.5 ml of the substrate, followed by adding 0.5 ml of CFS. A 10-min enzymatic reaction was carried out at 37 °C. Blank was prepared without addition of CFS. A final volume of 7.5 ml of 95% ethanol was added to terminate the reaction. Phenolphthalein indicator was used to titrate the released fatty acids against 0.1 M NaOH. The quantity of lipase that might release 1 µmol of fatty acids was measured in units of lipase activity. The mole of fatty acids was equal to the mole of the titrant and 1 mol NaOH was equal to 106 × µmol fatty acid equivalent. Lipase activity was determined using the Eqs. 2 and 3:   Eq. 2 Eq. 3 2.7. Esterase activity Colorimetric method was used to determine the esterase activity using modified method [19, 20]. Prior to esterase assessment, an overnight culture (24 h) of each strain was grown in modified Gibbon’s media containing 20% of NaCl, 2.5% of fish oil, 0.1% of MgSO4.7H2O, 0.3% of Na3C6H5O, 0.2% of KCl, 0.75% of tryptone and 0.1% of yeast extract for 3 d at 30 °C and 120 rpm. The CFS was used for the analysis. A mixture of 14 mM p-nitrophenyl butyrate in acetonitrile, 66.67 mM of Tris HCl (pH 8) and 3M of NaCl at a ratio of 1:8:1 was prepared as the substrate for the esterase activity assay. A volume of 20 µl of CFS and 200 µl of substrate were transferred into 96-well microplates and gently agitated. Double-distilled water was used as blank instead of CFS. Absorbance reading was carried out at 410 nm every 30 s at 37 °C over 5 min to measure the p-nitrophenol (p-NP) release using microplate reader (Powerwave X 340 microplate scanning spectro-photometer, BIO-TEK Instruments, Vermont, USA). Extinction coefficient (ε) for p-NP of this assay was 8222.1 M-1 cm-1, which was derived from the slope of the p-NP standard curve under similar detection conditions. For one unit (U) of esterase activity, enzyme quantity needed to release 1 µM of p-NP per minute was calculated based on Eq. 4 and Beer-Lambert Law as follows:                                                                                                                                                                                                                                Eq. 4 Where, ΔAbs showed changes in absorbance over time; ε denoted the molar extinction coefficient in M-1 cm-1; volume of assay represented the total volume of reaction mixture (ml); and Δt represented the incubation time (min). 2.8. Glutamic acid assessment Prior to assessment, the overnight culture of each strain was inoculated (1% v/v) into the glutamic acid production media (pH 7). This medium consisted of 5% of glucose, 0.8% of urea, 0.0002% of biotin, 0.1% of K2HPO4, 0.25% of MgSO4.7H2O, 0.01% of MnSO4.7H2O and 0.16% of CaCO3. Incubation was carried out at 30 °C for 7 d at 120 rpm. The crude glutamic acid source was the CFS that separated following centrifugation at 10,000 rpm for 10 min at 4 °C [21]. Glutamic acid was determined using glutamic acid colorimetric assay kit (Elabscience Biotechnology, Texas, USA). 2.9. Genome-based identification of the isolates Extraction of the bacterial DNA was carried out using Nucleospin microbial DNA extraction kit (Machery-Nagel, Duren, Germany). One loop of a single bacterial colony was pelleted and transferred to a microcentrifuge tube containing 40–400 μm of glass beads, added with buffer and proteinase and processed with FastPrep-24 system (MP Biomedicals, Ohio, USA). Lysed cells were centrifuged at 11,000× g for 30 s and DNA in the supernatant was purified using microbial DNA column based on the manufacturer’s protocol. Genome-based identification of the strains was carried out using Patriot Biotech, Selangor, Malaysia, based on the manufacturer’s standard protocol. Primers of 27F (TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG) and 1492R (ACTTGCCTGTCGCTCTAT-CTTCTACGGYTACCTTGTTACGACTT) with a Nano-pore partial adapter on the 5' end were used to amplify 16S rRNA full-length sequence of the strains. The PCR was carried out using WizBio HotStart 2× mastermix (Wizbiosolutions, Gyeonggi-do, Korea) with the following conditions of an initial denaturation step at 95 °C for 3 min; followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 50 °C for 20 s and extension at 72 °C for 120 s. The PCR products were visualized on agarose gels and purified using solid-phase reversible immobilization beads. Nanopore Flongle flow cell (Oxford Nanopore Technol-ogies, Oxford, UK) was used for the 24-h sequencing process. Sequences were compared to known sequences using BLAST online tool of the National Centre for Biotechnology (NCBI) website to verify the homology. The neighbor-joining trees were then constructed using MEGA 11 software. The rRNA sequence data of the selected strains were submitted to GenBank database. 2.10. Statistical analysis Minitab 19 statistical package (Minitab, Pennsylvania, USA) was used for the analysis. Results of the ANOVA analysis was reported statistically significant when p < 0.05. One-way ANOVA was carried out on variables to detect significant differences between the samples. Four-way ANOVA was used to assess effects of the multiple factors (time, temperature, NaCl concentration and strain) on the protease and lipase activities. To find out which groups of the samples were distinct from others, post hoc Tukey's honest significant difference (HSD) test was carried out. 2.11. Ethical considerations No experiments involving humans or animals were carried out in this study. Results and Discussion 3.1. Initial screening and isolation of proteolytic and lipolytic halotolerant bacteria For selecting bacteria for budu starter cultures, strains were initially assessed based on their ability to persist high-salt concentrations associated with budu fermentation. A total of 50 bacterial isolates were capable of growing on isolation media supplemented with 10–30% of NaCl (w/v) (data not shown). Twenty-nine isolates were originated from 12-m samples, eight from 6-m samples and 13 from 3-m samples. Only five isolates were recovered from NA containing 30% of NaCl, while 40 isolates were recovered from MA (20% NaCl). No colonies were observed in the MRSA containing 20% of NaCl, whereas, a few staphylococci were detected on MSA with 10% of NaCl. Decreased number of strains recovered from budu when grown on high-salt media could be due to the inactivation of key enzymes for microbial metabolism and growth. Additi-onally, the osmotic stress and metabolic adaptation forced bacteria to allocate their energy for adaptation rather than multiplication. Under further severe conditions, bacteria might enter viable but nonculturable (VBNC) states [22]. In a previous study, LAB and halophilic bacteria were isolated from Vietnamese fish sauces using salt concentrations ranging 5-18 and 18-25%, respectively. No growth of colonies was detected in high-salt media (approximately 29% NaCl) [10]. In the present study, strains capable of growing at 30% NaCl could be recovered; however, a longer incubation time of 14 d was needed for the colonies to become visible in the media, in contrast to the growth on MA that occurred within 72 h. Presence of LAB has been reported in Thai and

1-aminocyclopropane-1-carboxylate oxidase 2 reduction effects on physical and physiological responses of Eksotika papaya

The Malaysian Eksotika papaya (Carica papaya L.) has poor keeping quality due to its fast ripening attribute, which leads to post-harvest losses. This study is aimed at extending the shelf life of this perishable Eksotika fruit using antisense technology. A total of 6,000 Eksotika somatic embryogenic calli was transformed with the antisense 1-aminocyclopropane-1-carboxylic acid oxidase 2 gene (ACO2) construct, and 46 PCR-positive putative transformants were obtained. Gene expression analysis using real-time PCR on the 46 regenerated putative transgenic lines revealed that 42 showed down-regulation of the ACO2 gene with two- to five-fold differences among the lines. Out of 22 independently selected transformed lines grown under net house conditions, 16 harbored a single copy of the transgene. Physical stature of the transgenic plants was not significantly different from that of the non-transformed seed-derived papaya plants. Physiological evaluations of the transgenic fruits showed a 15-day delay in ripening compared with 4 days of the non-transformed seed-derived papaya fruits. The total soluble solid (TSS) of the transgenic fruits was comparable to that of the non-transformed seed-derived fruits with similar 11-15°Brix, implying the transgenes did not affect the TSS content

Comparative analysis of metabolites and antioxidant potentials from different plant parts of Curcuma aeruginosa roxb

A comparative analysis of metabolites from different parts of Curcuma aeruginosa, i.e. leaves, stems, adventitious roots and rhizomes was performed by GC-MS/MS coupled with multivariate statistical analysis. The GC-MS/MS analysis confirmed the occurrence of 26 metabolites belonged to terpenoids in almost all the samples. The Principal Component Analysis (PCA) indicated that there was a clear distinction between rhizomes and other plant parts, i.e. stems, leaves, and adventitious roots that could be explained by relatively higher contents of terpenoids including curzerene, alpha-farnesen, furanocoumarin, velleral, germacrone cineole, borneol, beta- and gamma- elemene and methenolone. The results of Hierarchical Clustering Analyses (HCA) corresponded with the PCA results where many terpenoids found abundantly high in rhizome were clustered together. This was supported by the Pearson correlation analysis that showed a significantly good relationship between those terpenoids. The adventitious roots demonstrated the strongest antioxidant activity as compared to the other plant parts which could be attributed to its highest Total Phenolic Contents (TPC). Total phenolic contents of all the plant parts were positively correlated with their antioxidant activities which indicate that phenolic compounds may play a role in the overall antioxidant activities of the plants. The results of the study highlighted the potential of this underexploited Curcuma species which could serve as a new source of important phytochemicals and natural antioxidant that could be incorporated in functional foods and nutraceuticals. In addition, chemical and biological evidence shown in the present work has rationalised the different uses of various plant parts of C. aeruginosa

Discovery of Functional SNPs via Genome-Wide Exploration of Malaysian Pigmented Rice Varieties

Recently, rice breeding program has shown increased interests on the pigmented rice varieties due to their benefits to human health. However, the genetic variation of pigmented rice varieties is still scarce and remains unexplored. Hence, we performed genome-wide SNP analysis from the genome resequencing of four Malaysian pigmented rice varieties, representing two black and two red rice varieties. The genome of four pigmented varieties was mapped against Nipponbare reference genome sequences, and 1.9 million SNPs were discovered. Of these, 622 SNPs with polymorphic sites were identified in 258 protein-coding genes related to metabolism, stress response, and transporter. Comparative analysis of 622 SNPs with polymorphic sites against six rice SNP datasets from the Ensembl Plants variation database was performed, and 70 SNPs were identified as novel SNPs. Analysis of SNPs in the flavonoid biosynthetic genes revealed 40 nonsynonymous SNPs, which has potential as molecular markers for rice seed colour identification. The highlighted SNPs in this study show effort in producing valuable genomic resources for application in the rice breeding program, towards the genetic improvement of new and improved pigmented rice varieties

Bacterial community structure, predicted metabolic activities, and formation of volatile compounds attributed to Malaysian fish sauce flavour

The fermentation of Malaysian fish sauce (budu) varies from one to twelve months depending on the producer, resulting in inconsistent quality. The microbiota, their predicted metabolic pathways and volatile metabolites profiles were determined at different stages of budu fermentation. Budu fermented for 1 and 3 months were characterized by the presence of Gram negative Enterobacterales, Gammaproteobacteria, and Fusobacteriaceae, which continuously decrease in abundance over fermentation time. The metabolic pathways prediction grouped 1- and 3- month budu in a cluster enriched with degradation reactions. 6-month budu were dominated by Halanaerobium and Staphylococcus, while the 12-month were dominated by Lentibacillus, Bacilli, and Halomonas. Biosynthesis-type predicted pathways involving protein and lipid derivatives were enriched in 6- and 12-month fermented budu, accumulating 2,6-dimethylpyrazine, methyl 2-ethyldecanoate, 2-phenylacetaldehyde, 3-methylbutanal, and 3-methylbutanoic acid. These compounds may indicate budu maturity and quality. This result may assist as a reference for quality control and fermentation monitoring

Overexpression of type II rice metacaspase, OsMC4, increases endoplasmic reticulum stress tolerance in transgenic rice calli

The endoplasmic reticulum (ER) is an organelle responsible for regulating protein synthesis in plants. High salinity can lead to the accumulation of misfolded proteins, resulting in an ER stress response mechanism known as the unfolded protein response (UPR). Failure of the UPR to reverse the effect of protein misfolding will activate programmed cell death (PCD). Metacaspase genes are known regulate PCD in plants. This study aims to provide a comprehensive analysis of the expression patterns of type II rice metacaspase (OsMC) genes in response to ER and salinity stress in rice leaf. Among five type II metacaspases in the rice genome, OsMC4, OsMC5, and OsMC8 expressions were found to be upregulated during treatment with tunicamycin (ER stress) and sodium chloride, NaCl (salinity stress). A construct of taqRFP::OsMC4, controlled by the CaMV35S promoter, was generated and transformed into rice calli. The transgenic rice calli overexpressing taqRFP::OsMC4 demonstrated significant changes in the expression of the ER stress-marker genes, protein disulfide isomerase (OsPDI), and binding immunoglobulin protein (OsBiP). The results from this analysis provide preliminary evidence that at least one of the type II metacaspases, OsMC4, is be able to reduce ER and salinity stresses in rice. Further functional analysis of OsMC genes in ER and salinity stress tolerance could be carried out in transgenic rice overexpressing OsMCs in the future to improve stress tolerance. Simple summary: Oryza sativa, commonly known as rice, is one of the most consumed crops in the world. In response to multiple biotic and abiotic factors, a series of endoplasmic reticulum (ER) stress response regulators are activated. There is evidence that high salinity triggers ER stress in plants. This study aims to determine the level of gene expression among type II metacaspases in rice in response to ER and salinity stress and to assess how they may be linked to PCD in rice calli. Three metacaspase genes, OsMC4, OsMC5, and OsMC8, have been observed to have significant expression post-treatment with tunicamycin in rice leaf. Overexpression of taqRFP::OsMC4 in rice calli significantly reduces the expression level of the stress markers, OsBiP and OsPDI, indicating that the stress level is relatively lower in the transgenic calli compared to the wild-type calli. Therefore, overexpression of taqRFP::OsMC4 in rice may increase rice tolerance towards ER and salinity stress. These expression analyses of the OsMCs family provide valuable information for further functional studies on the biological roles of OsMCs in ER and salinity stress responses.</p