Phenotypic and molecular characterisations of lactic acid bacteria isolated from Malaysian fruits

Lactic Acid Bacteria (LAB) are gram-positive, catalase-negative and non-spore forming bacteria known to have many advantages such as starter culture in food fermentation, as antimicrobial agent and plant growth promoter. Limited information on various LAB present in Malaysian fruits hampers further study to explore their potential as autochthonous inoculants in food fermentation, plant disease control and growth promotion. Therefore, the objectives of this study are firstly to isolate and identify LAB from honeydew, ciku, mango and mata kucing by investigating their morphological and biochemical characteristics, secondly to determine the identity of the isolates using 16S rRNA gene sequencing and finally to examine phylogenetic relationship of the LAB present in the fruits. The isolates were subjected to Gram staining, acidity and catalase tests, followed by molecular identification and phylogenetic analysis of the bacteria. Out of 33 isolates, eight isolates were gram- positive, catalase-negative and acid producers, suggesting that they are potentially LAB. 16S rRNA sequencing and NCBI Blast analysis identified the presence of Lactococcus sp., Leuconostoc sp., Weissella sp. and Aerococcus sp. in the fruit samples with sequence identity 94-97%. Phylogenetic tree was constructed based on the 16S rRNA sequences using Neighbor-Joining method. This study has assisted in collecting more information about the diversity of LAB in Malaysian fruits, which can be further explored in future for their application as bioinoculant in food fermentation or as biocontrol agent and plant growth promoter in agricultural field

Analysis of bacterial communities in rhizosphere soil of symptomless and basal stem rot (BSR)-infected oil palm using terminal restriction fragment length polymorphism (T-RFLP)

Basal Stem Rot (BSR) disease caused by pathogenic fungi known as Ganoderma boninense has been identified as a major threat in oil palm plantation. Previously used methods to control this disease have been ineffective while method using chemical treatment is not environmentally friendly. An inadequate knowledge on the core microbiome of oil palm rhizosphere and the relationship between BSR disease incidences hinders effective controls against this pathogenic disease. Hence, the objectives of this project are to determine the bacterial communities of symptomless and BSR-infected oil palm using T-RFLP analysis, to perform cluster analysis of the samples based on the T-RFLP data and to analyze the relationship between soil bacterial communities and soil pH. The rhizosphere samples of symptomless and BSR-infected oil palm were collected at different microsites (bulk soil from harvesting path and rhizosphere soil from weeded circle and frond pile) and at different depths (10 cm and 30 cm from upper soil surface) from Oil Palm Plantation, Seberang Perak. In T-RFLP analysis, 16s rRNA region of the bacterial DNA were amplified by using 8F forward primer labelled with 6-FAM fluorescent dye and unlabelled 1492R reverse primer. The PCR products were then digested either with restriction enzyme AluI or HhaI or double digestion using AluI and HhaI. The raw fragments data were aligned and analyzed in T-REX (T-RFLP Analysis Expedited Software). The ordination analysis of Additive Main Effects and Multiplicative Interaction Model (AMMI) analysis from T-REX software revealed higher percentages of signal compared to noise interaction effects for frond pile at depth 30 cm with interaction difference of 21.28% (analysis based on relative abundance). The higher difference between signal and noise indicates that there are larger differences in microbial community between symptomless and BSR-infected oil palm. Meanwhile, cluster analysis showed that the sample obtained from harvesting path (bulk soil) at 10 cm and 30 cm depth clustered closely together indicating that there are small differences in microbial community at these microsites. Clustering analysis based on relative abundance shows that there is larger difference in microbial abundance between symptomless and BSR-infected sample at Frond Pile (30 cm depth). These results will provide preliminary knowledge in selecting representative samples of symptomless and BSR-infected oil palm for further microbial interaction analysis using Next Generation Sequencing

EFFECTIVENESS OF THE FERMENTATIVE EXTRACT OF Lactobacillus acidophilus AS ANTIMICROBIALS AGAINST Aeromonas hydrophila

The purpose of this study was to evaluate the in vitro and in vivo antimicrobial activity of the commercial Lactobacillus acidophilus (L. acidophilus) cells and cell free extract against Aeromonas hydrophila (A. hydrophila). The in vitro method was carried out using well diffusion method. For in vivo evaluation, the effect of L. acidophilus on the survival rate of Pangasianodon hypophthalmus (P. hypophthalmus) infected with A. hydrophila was evaluated. The well diffusion method showed a significant inhibition ability of L. acidophilus cells against A. hydrophila compared to the cell free extract. The inhibition diameters obtained with cells and cell free extract were 17.23 mm and 15.17 mm, respectively. P. hypophthalmus injected with L. acidophilus cells and cell free extract following challenged with A. hydrophila cells showed survival rate of 70% and 60% respectively, at 2-week post challenged. The gas chromatography-mass spectrophotometry (GC-MS) result revealed that a diverse of compounds was detected in both the L. acidophilus cells and cell free extract, among them the most abundant component was pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), which showed a promising anticancerous activity and might be played a significant role in the recovery of the infectious P. hypophthalmus. The current study revealed that both cells and cell free extract of L. acidophilus have antimicrobial activity against A. hydrophila

Soil pH analysis in relation to basal system rot (BSR) disease in oil palm at different depths and microsites

In agricultural industry, soil is known as important indicator for plant growth and productivity. Variations in soil conditions will alter soil ecosystem and communities that exist within the environment. Significant changes in soil environment sometimes favor the growth of plant pathogen and thus affecting plant health. Basal Stem Rot (BSR) disease caused by pathogenic fungi known as Ganoderma boninense has been identified as a major threat in oil palm plantation. The establishment of different microsites (weeded circle, harvesting path and frond pile) in oil palm management may induce the spatial differences of soil properties within a field including soil pH. Hence, our aim is to study the adverse changes in soil pH in relation to BSR disease of oil palm at different microsites (harvesting path, weeded circle and frond pile) and soil depth (10 cm and 30 cm from upper soil surface). In general, the soil samples of oil palm obtained from FELCRA Seberang Perak have shown acidic nature in which soil from infected oil palm has lower pH value (pH range 2.93-5.94) as compared to soil pH from healthy oil palm (pH range 3.39-6.29). The statistical results showed that soil depth has no significant effect to the soil pH while soil from different microsites has a significant effect to the soil pH regardless of healthy or infected oil palm. At different microsites, weeded circle give the most acidic value as compared to harvesting path and frond pile. Besides, the interaction between healthiness, depth and microsites did not have a significant effect to the soil pH. Together, these results suggest that BSR disease are likely to occur at soil with lower pH (pH below 6) and soil pH are strongly correlates with different microsites but less affected by soil depth regardless of the healthiness of oil palm

Collagen binding activity of bacteria isolated from pig and cow small intestine

Bacterial adherence to connective tissue, especially to collagen has been vastly known for their invasive and infectious activities. However, the ability to exploit the unique and specific interactions between bacteria and collagen as a novel approach in detection of placental collagen has never been explored. This study aimed to determine bacteria with binding specificity to placental collagen (Type IV) derived from human and sheep. In order to do this, total bacteria from small intestines of pig and cow were isolated and their ability to bind to Type IV placental collagen (human and sheep) was determined. Interestingly, three bacterial samples; P5, P9 (pig small intestine origin) and B7 (cow small intestine origin) were found to be able to bind strongly to the placental collagen. The bacterial binding to human placental collagen was however, diminished after the bacteria were treated with trypsin, proteinase K (for removal of surface protein) and guanidine hydrochloride (for S-layer removal), suggesting that the interaction of these bacteria to placental collagen was promoted by protein(s) present at the bacterial surface. In addition, significant reduction of placental collagen-binding ability of the bacteria pre-incubated with soluble human placental collagen showed that there is a specific interaction between the bacteria and collagen. P5, P9 and B7 bacteria were found to share 95-97% 16S rRNA sequence similarity to Enterococcus faecalis ZL, Enterococcus hirae ss33b and Enterococcus faecium M3-1, respectively. The results presented here may facilitate future studies in identifying bacterial surface protein(s) responsible for the specific binding of bacteria to collagen and opens new opportunity to utilize the protein(s) for the detection of placental collagen in nutraceutical and food supplements

In vitro studies of lactic acid bacteria against the causative agent of papaya dieback disease

Papaya is an economically significant fruit crop grown in Malaysia. Nevertheless, the outbreak of Papaya Dieback disease has caused major threats to the papaya plantation for almost a decade as effective treatment found to date has been limited. Endophytic bacteria have been used as biological control agent against several plant diseases. This study proposed that bacterial endophytes isolated from papaya plant might be used as biological alternative to synthetic bactericide to restrain this disease. The aim of this study is to find an effective microorganism to suppress Papaya Dieback disease. In this study, a total of 230 bacterial endophytes with antagonistic activities against Erwinia mallotivora were isolated from seeds and sarcotesta of two papayas (Carica papaya L.) collected from Sabak Bernam (PPS) and Perak (PPK) through rapid screening using Agar Overlay method. Twenty-eight pure isolates from the respective PPS and PPK showed significant inhibitory effect against Erwinia mallotivora as revealed by Agar Disc Diffusion technique. PPKSD19 showed the highest value of inhibition zone at 21.7 mm during in vitro screening on MRS agar. The isolates were characterized as Gram-positive rods, cocci or coccobacilli, catalase - negative and positive in acidity test, suggesting that they are potentially Lactic Acid Bacteria. API 50 CH profiles and 16S rRNA sequencing allowed identification of bacteria as Weissella sp. and Lactococcus sp. The selected high-performing endophytic bacteria will be investigated for their synergistic activities and bacteriocin-production. Their effects on infected papaya plant will be tested under greenhouse experiment. Our findings suggest that Carica papaya L. seed-borne bacterial endophytes could potentially be applied as biological control agent to inhibit Papaya Dieback disease

Improved gel-enhanced liquid chromatography-mass spectrometry by chemometrics for halal proteomics

Numerous analytical methods for the authentication of halal meat are now well established, with gel-enhanced liquid chromatography-mass spectrometry (GeLCMS) being a popular approach. However, the selection of potential protein markers on 1-dimensional gel electrophoresis (1DE) prior to LCMS is considered problematic, because using the optical density for the selection process could introduce human error. In this study, an improved GeLCMS method assisted by multivariate principal component analysis (PCA) was developed to identify the potential protein markers for non-halal pork among halal beef and chicken. The improved GeLCMS technique allowed for the confident excising of identified protein bands prior to in-gel tryptic digestion. The inferential protein markers (myofibrillar proteins), which might be present in the samples, were determined based on the identified sequence of peptides. This chemometric-assisted GeLCMS could potentially be used as a guideline to assist chemists in analysis of any gel-based separation of biomolecules, regardless of the field of stud

Evaluation of toxicity effect of palm oil mill effluent final discharge by using Daphnia magna

Palm Oil Mill Effluent (POME) final discharge has a risk to the ecosystem due to various harmful contaminants including organic and inorganic materials. In this study, biological monitoring method was used to evaluate the toxicity effects of POME final discharge using Daphnia magna. The physical and chemical nature of toxicants present in the effluent were characterized through acute Whole Effluent Toxicity (WET), and Toxicity Identification Evaluation (TIE) tests. The Toxicity Unit (TU) and median lethal concentration (LC50) of the POME sample were 11.09 and 9.02% (v/v) respectively. From TIE test, the toxicants present in the effluent can be characterized as filterable and oxidisable through filtration and aeration treatment. The presence of cationic metals, chlorine and disinfection by-products were also determined by the toxicity reduction of the effluent after treatment using ethylenediaminetetraacetic acid (EDTA) and sodium thiosulphate. From TIE test, the filtration treatment at pH 10 of the POME final discharge was the most effective method in reducing the toxicity of the effluent with a value of TU, 1.16 and LC50, 86.34% (v/v). It is recommended that biological tests using Daphnia magna can be made as potential methods to indicate the effects of POME final discharge to the aquatic ecosystem

Alteration of the Gut Microbiome in Normal and Overweight School Children from Selangor with \u3ci\u3eLactobacillus\u3c/i\u3e Fermented Milk Administration

Childhood obesity is a serious public health problem worldwide. Perturbations in the gut microbiota composition have been associated with the development of obesity in both children and adults. Probiotics, on the other hand, are proven to restore the composition of the gut microbiome which helps reduce the development of obesity. However, data on the effect of probiotics on gut microbiota and its association with childhood obesity is limited. This study aims to determine the effect of probiotics supplement intervention on gut microbiota profiles in obese and normal-weight children. A total of 37 children, 17 normal weight, and 20 overweight school children from a government school in Selangor were selected to participate in this study. Participants were further divided into intervention and control groups. The intervention groups received daily probiotic drinks while the control groups continued eating their typical diet. Fecal samples were collected from the participants for DNA extraction. The hypervariable V3 and V4 regions of 16S rRNA gene were amplified and sequenced using the Illumina MiSeq platform. No significant differences in alpha diversity were observed between normal weight and obese children in terms of the Shannon Index for evenness or species richness. However, a higher intervention effect on alpha diversity was observed among normal-weight participants compared to obese. The participants’ microbiome was found to fluctuate throughout the study. Analysis of the taxa at species level showed an increase in Bacteroides ovatus among the normal weight cohort. Genus-level comparison revealed a rise in genus Lachnospira and Ruminococcus in the overweight participants after intervention, compared to the normal-weight participants. The probiotics intervention causes an alteration in gut microbiota composition in both normal and overweight children. Though the association could not be defined statistically, this study has provided an improved understanding of the intervention effect of probiotics on gut microbiome dysbiosis in an underrepresented population

Comparing the effect of heat on tropomyosin isoforms patterns from water buffalo and wild boar meat by two-dimensional gel electrophoresis

Tropomyosin is one of the most abundant proteins in meat; however, very little is known about it due to the lack of scientific literature. In this study, the spot volume of tropomyosin (TPM) isoforms, TPM2 and TPM1, in meat from water buffalo and wild boar subjected to various cook treatments were compared. We hypothesized that primary structures of the tropomyosin isoforms from both species would remain stable despite the application of heat. Proteins extracted from the treated meats were analyzed using two-dimensional gel electrophoresis and mass spectrometry. A Kruskal-Wallis test showed that there were no significant differences in protein spot volumes for all treatments; however, a significant difference was observed between species. Changes in the amino acid sequence of TPM1 were observed between the two species, indicating that the isoforms could be used as thermostable proteins or peptide markers for species identification because of their resistance to high temperatures