The biodiversity and the killer activity of yeasts isolated from various types of fermented food in Malaysia were investigated in this study. Of 252 yeasts isolated from 48 fermented food samples in this study, 19 yeast species were identified based on sequence analysis of the ITS1-5.8S-ITS2 partial fragments of the yeasts. A total of 29 (11.5%) of the yeast isolates demonstrated killer activity to at least one Candida species tested in this study; including 22 isolates of Trichosporon asahii, 4 isolates of Pichia anomala, and one isolate each of Pichia norvegensis, Pichia fermentans and Issatchenkia orientalis, respectively. The presence of killer yeasts reflects antagonism that occurs during microbial interaction in the fermented food, whereby certain yeasts produce killer toxins and possibly other toxic substances in competition for limited nutrients and space. The anti-Candida activity demonstrated by killer yeasts in this study should be further explored for development of alternative therapy against candidiasis.
This study describes the killer phenotypes of tropical environmental yeasts and the inhibition effects of the culture filtrates on the biofilm of Candida albicans. A total of 26 (10.5%) of 258 yeast isolates obtained from an environmental sampling study demonstrated killer activity to Candida species. The killer yeasts were identified as species belonging to the genus Aureobasidium, Pseudozyma, Ustilago and Candida based on sequence analysis of the ITS1-5.8S-ITS2 region of the yeasts. Pseudozyma showed the broadest killing effects against sensitive strains of Candida. New species of Ustilago and Pseudozyma demonstrating killer phenotypes were identified in this study. Interestingly, more than 50% reduction in the metabolic activity of Candida albicans biofilm was noted after exposure to the culture filtrates of the nine killer yeasts. Purification and characterization of toxin and metabolites are essential for understanding the yeast killing effects.
Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.
Mixotrophic metabolism was evaluated as an option to augment the growth and lipid production of marine microalga Tetraselmis sp. FTC 209. In this study, a five-level three-factor central composite design (CCD) was implemented in order to enrich the W-30 algal growth medium. Response surface methodology (RSM) was employed to model the effect of three medium variables, that is, glucose (organic C source), NaNO3 (primary N source), and yeast extract (supplementary N, amino acids, and vitamins) on biomass concentration, X(max), and lipid yield, P(max)/X(max). RSM capability was also weighed against an artificial neural network (ANN) approach for predicting a composition that would result in maximum lipid productivity, Pr(lipid). A quadratic regression from RSM and a Levenberg-Marquardt trained ANN network composed of 10 hidden neurons eventually produced comparable results, albeit ANN formulation was observed to yield higher values of response outputs. Finalized glucose (24.05 g/L), NaNO3 (4.70 g/L), and yeast extract (0.93 g/L) concentration, affected an increase of X(max) to 12.38 g/L and lipid a accumulation of 195.77 mg/g dcw. This contributed to a lipid productivity of 173.11 mg/L per day in the course of two-week cultivation.
Vaginal discharge is a common complaint of women attending gynaecological clinics. The purpose of this study was to compare the occurrence of commonly implicated microorganisms in vaginal discharge amongst women with or without the complaint, attending a gynaecological and family planning clinic. The association of Gardnerella vaginalis with bacterial vaginosis was also studied. It was found that there were no significant differences between the cases and controls in the isolation rate of Gardnerella vaginalis, Torulopsis glabrata, Ureaplasma urealyticum, Mycoplasma ssp and Group B streptococcus (p greater than 0.05). Only the isolation rate of Candida albicans was significantly higher in the cases than controls (p less than 0.01). However, there was a significant association of G. vaginalis with bacterial vaginosis.
The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.
Prevalence of dermatomycoses varies from one centre to another due to many factors. Knowledge of local prevalence is useful to aid clinical diagnosis and treatment. Due to lack of data in Malaysia, this study aimed to look at the causes of dermatomycoses in Kuala Lumpur, Malaysia. Dermatological specimens including skin scrapings, hair and nail clippings were collected carefully from clinically suspected cases of dermatomycoses between 2008 and 2010. All cultures of skin, hair and nails that yielded positive fungal growth were included. Any fungal growth outside the streaking area, duplicate and incomplete data were excluded from the study. Three-hundred-fifty-eight patients were included. Male patients were slightly more than females with a ratio of 1.2:1. The median age was 53 years old with interquartile range of 38-64 years. More than half (53.6%) belonged to 20-60 years age group. Rates of culture isolation were 89.0% for nails, 56.2% for hair and 55.6% for skin. Five-hundred-twenty-two fungi were isolated from 358 clinical specimens. Non-dermatophyte moulds (NDMs) represented the largest group (50.5%; mainly Aspergillus species 18.7%), followed by yeasts (41.6%; mainly Candida species 26.8%) and dermatophytes (7.9%; mainly Trichophyton species 7.7%). In conclusion, NDMs and yeasts were more commonly isolated than dermatophytes from dermatological specimens in this centre. Current treatment regime that focuses on dermatophytes may be ineffective to treat dermatomycoses caused by NDMs or yeasts. Antifungal susceptibility study may be needed to guide therapy in recalcitrant cases.
Onychomycosis is the infection of nail apparatus by dermatophytes, yeasts or non-dermatophyte moulds and is responsible for 50% of all nail disorders. A five year retrospective study was conducted at Universiti Kebangsaan Malaysia to identify the common pathogens responsible for onychomycosis and to describe the epidemiology of the affected patients. A total of 278 abnormal nails were cultured, out of which 231 were positive for fungus. Females constituted 50.2% (n=116) while males 49.8% (n=115). The majority (51.9%, n=120) were between ages 50-69 years. The Malay ethnic group was most commonly affected (44.2%, n=102) followed by Chinese (33.8%, n=78), Indians (18.2%, n=42) and other ethnic groups (3.8%, n=9). The most common fungal element isolated was non-dermatophyte moulds (45.4%, n=105) followed by yeast (34.6%, n=80) and dermatophytes (1.3%, n=3). Aspergillus spp. was the commonest (59.8%,n=81) non-dermatophyte mould, while Candida spp. was the commonest yeast (74.3%, n=89) isolated. In this study, non-dermatophyte moulds are the most common microorganisms implicated to cause onychomysosis. Treatment for non-dermatophyte mould is challenging as the current available antifungal agents are more effective against dermatophytes and yeasts.
Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.
Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at micro = 0.396 h-1 and ST3 at micro = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (STI and ST3) to produce bioethanol via immobilization technique with local materials as substrate.
A total of 2,153 high vaginal swabs were processed for the presence of yeasts. The specimens were obtained from pregnant and non-pregnant Malaysian women with and without vaginitis. The yeast species most commonly isolated were Candida albicans, C. glabrata, C. famata and C. parapsilosis. C. albicans was isolated from 27% of pregnant women with vaginitis, 14% of pregnant women with no overt vaginitis, 15% of non-pregnant women with vaginitis, and 3% of non-pregnant women with no vaginitis. The significant difference of the isolation rates from women with and without vaginitis indicates that C. albicans is likely to be a vaginal pathogen.
A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.
The production of lipids from oleaginous yeasts involves several stages starting from cultivation and lipid accumulation, biomass harvesting and finally lipids extraction. However, the complex and relatively resistant cell wall of yeasts limits the full recovery of intracellular lipids and usually solvent extraction is not sufficient to effectively extract the lipid bodies. A pretreatment or cell disruption method is hence a prerequisite prior to solvent extraction. In general, there are no recovery methods that are equally efficient for different species of oleaginous yeasts. Each method adopts different mechanisms to disrupt cells and extract the lipids, thus a systematic evaluation is essential before choosing a particular method. In this review, mechanical (bead mill, ultrasonication, homogenization and microwave) and nonmechanical (enzyme, acid, base digestions and osmotic shock) methods that are currently used for the disruption or permeabilization of oleaginous yeasts are discussed based on their principle, application and feasibility, including their effects on the lipid yield. The attempts of using conventional and "green" solvents to selectively extract lipids are compared. Other emerging methods such as automated pressurized liquid extraction, supercritical fluid extraction and simultaneous in situ lipid recovery using capturing agents are also reviewed to facilitate the choice of more effective lipid recovery methods.
Chemical preservatives have been used in the food industry for many years. However, with increased health concerns, consumers prefer additive-free products or food preservatives based on natural products. This study evaluated antimicrobial activities of extracts from Emilia sonchifolia L. (Common name: lilac tassel flower), Tridax procumbens L. (Common name: tridax daisy) and Vernonia cinerea L. (Common name: Sahadevi), belonging to the Asteracea family, to explore their potential for use against general food spoilage and human pathogens so that new food preservatives may be developed. Three methanol extracts of these plants were tested in vitro against 20 bacterial species, 3 yeast species, and 12 filamentous fungi by the agar diffusion and broth dilution methods. The V. cinerea extract was found to be most effective against all of the tested organisms and the methanol fraction showed the most significant (p < 0.05) antimicrobial
activity among all the soluble fractions tested. The minimum inhibitory concentrations (MICs) of extracts determined by the broth dilution method ranged from 1.56 to 100.00mg/mL. The MIC of methanol fraction was the lowest in comparison to the other four extracts. The study findings indicate that bioactive natural products from these plants may be isolated for further testing as leads in the development of new pharmaceuticals in food preservation as well as natural plant-based medicine.
Ethanolic extract of Cassia alata leaves was investigated for its antimicrobial activities on several microorganisms including bacteria, yeast, dermatophytic fungi and non-dermatophytic fungi. In vitro, the extract exhibited high activity against various species of dermatophytic fungi but low activity against non-dermatophytic fungi. However, bacterial and yeast species showed resistance against in vitro treatment with the extract. The minimum inhibitory concentration (MIC) values of the extract revealed that Trichophyton mentagorphytes var. interdigitale, Trichophyton mentagrophytes var. mentagorophytes, Trichophyton rubrum and Microsporum gypseum had the MIC of 125 mg/ml, whereas Microsporum canis had the MIC of 62.5 mg/ml. The inhibition can be observed on the macroconidia of Microsporum gypseum which resulted in structural degeneration beyond repair. The mechanism of inhibition can be related to the cell leakage as observed by irregular, wrinkle shape and loss in rigidity of the macroconidia.
Copper nanoparticle synthesis has been gaining attention due to its availability. However, factors such as agglomeration and rapid oxidation have made it a difficult research area. In the present work, pure copper nanoparticles were prepared in the presence of a chitosan stabilizer through chemical means. The purity of the nanoparticles was authenticated using different characterization techniques, including ultraviolet visible spectroscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The antibacterial as well as antifungal activity of the nanoparticles were investigated using several microorganisms of interest, including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella choleraesuis, and Candida albicans. The effect of a chitosan medium on growth of the microorganism was studied, and this was found to influence growth rate. The size of the copper nanoparticles obtained was in the range of 2-350 nm, depending on the concentration of the chitosan stabilizer.
The yield and properties of cellulose produced from bacterial fermentation of black tea broth (known as Kombucha) were investigated in this study. The tea broth was fermented naturally over a period of up to 8 days in the presence of sucrose. Tea broth with a sucrose concentration of 90 g/l produced highest yield of bacterial cellulose (66.9%). The thickness and yield of bacterial cellulose increased with fermentation time. The bacterial cellulose production increased correspondingly with increased surface area:depth ratio. Changes in pH were related to the symbiotic metabolic activities of yeasts and acetic acid bacteria, and the counts of both of these in the tea broths were relatively higher than those in the cellulose layer. Findings from this study suggest that the yield of cellulose depends on many factors that need to be optimized to achieve maximum yield.
Cantaloupes continue to ripen after harvesting which is caused by ethylene production due to climacteric behaviour during postharvest storage. In this study, the cantaloupe fruits harvested at commercial maturity were evaluated for quality attributes during three weeks of storage at 10°C and a relative humidity (RH) of 90±5%. In addition, fresh-cut samples were stored for a further 19 days at 2°C and 87% RH. The fresh-cut samples were prepared on a weekly basis by dipping into deionised water (control) at 2°C for 1 minute. The effect of postharvest storage of cantaloupe on the physico-chemical properties and microbial activity was observed prior to fresh-cut processing. It was found that firmness, luminosity (L*), and titratable acidity (TA) decreased, while total soluble solids (TSS), pH, TSS:TA ratio, microbial activity (total plate count (TPC) and yeast and mould (YM)) of the fresh-cut increased over the postharvest storage period of the fruit. Meanwhile, the orange colour and the intensity (hue angle, hab, and chromaticity) of the flesh did not differ significantly during storage. The cantaloupe stored for three weeks at a low temperature indicated a successful potential for fresh-cut processing due to good maintenance of the product quality.
Bioprotein is one of the useful products obtained from biotechnology invention. It is a promising replacement for the commercial fish feed supplement. In this study, the enrichment of the bioprotein content after solid state fermentation using palm kernel cake and seaweed by the white rot fungus: Phanerochaete chrysoporium and yeast: Candida utilis was carried out. The growth media components were selected from 11 types of media using Plackett-Burman design (hereinafter PBD) and were optimized by one-factor-at-a-time (OFAT) method with bioprotein concentration (mg/g) as the response. From the screening result using PBD, three media components, namely K2HPO4, CuSO4.5H2O and MnSO4.H2O were selected for further optimization using OFAT method because of their positive contributions to the response. The final results showed that 5.0 g/L K2HPO4, 3.0 g/L CuSO4.5H2O and 0.1 g/L MnSO4.H2O were there to be the optimum media constituents with 9.0 g/L, MgSO4.7H2O, 0.1 g/L, CaCl2.H2O, 3.0 g/L FeSO4.7H2O and 3.0 g/L peptone as fixed compositions. At this optimum concentration, the protein increment of 11% was observed as compared to the results determined in the screening using PBD. The study revealed the benefits of using mixed cultures in improving the protein concentrations which can be used as nutritious fish feed.