Applications of FT-NIRS combined with PLS multivariate methods for the detection & quantification of saccharin adulteration incommercial fruit juices

<p>Detection of adulteration in carbohydrates rich foods like fruit juices is particularly difficult because of the variety of the commercial sweeteners available that match the concentration profiles of the major carbohydrates in the foods. In present study, a new sensitive and robust assay using Fourier Transform Near Infrared Spectroscopy (FT-NIRS) combined with partial least square (PLS) multivariate methods has been developed for detection and quantification of saccharin adulteration in different commercial fruit juice samples. For this investigation 6 different commercially available fruit juice samples were intentionally-adulterated with saccharin at the following percentage levels: 0 %, 0.10 %, 0.30 %, 0.50 %, 0.70 %, 0.90 %, 1.10 %, 1.30 %, 1.50 %, 1.70 % and 2.00 % (weight/volume). Altogether 198 samples were used including 18 pure juice samples (un-adulterated) and 180 juice samples adulterated with saccharin. PLS multivariate methods including partial least-squares discriminant analysis (PLS-DA), and partial least-squares regressions (PLSR) were applied to the obtained spectral data to build models. The PLSDA model was employed to differentiate between pure fruit juice samples and those adulterated with saccharin. The R² value obtained for the PLS-DA model was 97.90 % with an RMSE error of 0.67 %. Similarly, a PLS regression model was also developed to quantify the amount of saccharin adulterant in juice samples. The R² value obtained for the PLSR model was 97.04 % with RMSECV error of 0.88 %. The employed model was then cross-validated by using a test set which included 30 % of the total adulterated juice samples. The excellent performance of the model was proved by the low RMSEP value of 0.92 % and the high correlation factor of 0.97. This newly developed method is robust, non-destructive, highly sensitive, and economical.</p

Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid

<div><p><i>Boswellia sacra</i>, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%), Chaetomiaceae (17.6%), Incertae sadis (29.5%), Aureobasidiaceae (17.6%), Nectriaceae (5.9%) and Sporomiaceae (17.6%) from the phylloplane (leaf) and caulosphere (stem) of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33%) than the stem (0.262%). The Shannon-Weiner diversity index was <i>H</i>′ 0.8729, while Simpson index was higher in the leaf (0.583) than in the stem (0.416). Regarding the endophytic fungi’s potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, <i>Penicillum citrinum</i> BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 μM^-1min^-1mL) and cellulases (62.11±1.6 μM^-1min^-1mL), whereas <i>Preussia</i> sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 μM^-1min^-1mL) and phosphatases (3.46±0.31μM^-1min^-1mL) compared to other strains. <i>Aureobasidium</i> sp. BSS6 and <i>Preussia</i> sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways). <i>Preussia</i> sp. BSL10 was applied to the host <i>B</i>. <i>sacra</i> tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin could establish a unique niche for ecological adaptation during symbiosis with the host Frankincense tree.</p></div

Indole acetic acid (IAA) content in the culture filtrate of various endophytic fungal strains.

<p>L-tryptophan-dependent Czapek media was used to grow fungal strains for 7 days. A standard in the same media was also read to yield a standard curve (R² = 0.9985). A total of five replications (100 ml in each Erlenmeyer flask) were used to make sure the validation of IAA results. The bars representing different letters show that the mean values are significantly different from each other, as evaluated by the DMRT test (<i>P<</i>0.05).</p

Endophytic fungal strains isolated from the stem and leaf parts of the Frankincense tree.

<p>Endophytic fungal strains isolated from the stem and leaf parts of the Frankincense tree.</p

Influence of endophyte inoculation on Host growth.

<p>Effect of the application of endophytic fungus (<i>Preussia sp</i>. BSL10) on the growth dynamics and photosynthetic pigments of <i>Boswellia sacra</i> tree saplings. Each treatment included nine replications. The photosynthetic pigments were measured after 21 DAT. The graphs show the mean value of three replications with standard deviations. 0 DAT shows the readings without any treatments applied.</p

Sample collection and processing for the isolation of fungal endophytes from the frankincense (<i>Boswellia sacra</i>) tree.

<p>(<b>A</b>) Shows the hot and dry habitat of the frankincense tree; (<b>B</b>) resin emerging from the mature leaf of tree branches; (<b>C</b>) the milk-like resin oozing out from the lower epidermal layer of the tree; (<b>D</b>) the inner bark or the cortex region of the tree did not contain resinous ducts and was also evaluated for the presence of endophytes; (<b>E</b>) dried wounded outer bark in which the white milk was converted into crystalline gum (<b>F</b>); leaf pattern of the frankincense tree; (<b>G</b>) smaller parts of the tree (leaf, stem and bark parts) were used for isolation. A total of 208 10-mm tissue samples from the leaf and stem (bark) were used to isolate endophytic fungi. The black line is equivalent to 1 inch.</p

Phylogenetic analysis of endophytes.

<p>Maximum Parsimony analysis of isolated endophytes based on the sequences obtained from the sequencing process of the internal transcribed spacer (ITS) region. The phylogeny was constructed using the homologous fungal sequences deposited in GenBank. The percentage of replicate trees in which the associated species clustered together in the bootstrap test (2000 replicates) are shown next to the branches. The analysis involved 84 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 296 positions in the final dataset. Evolutionary analyses were conducted in MEGA 6.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158207#pone.0158207.ref030" target="_blank">30</a>].</p

Endophytic distribution and nucleotide homology.

<p>Endophyte isolated from <i>B</i>. <i>sacra</i>, family-wise distribution (A) and comparison of nucleotide composition in the sequence with those from related strains in GenBank (B). A comparative assessment was made with the help of MEGA 6.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158207#pone.0158207.ref030" target="_blank">30</a>].</p

Principal Component Analysis (PCA) analysis.

<p>PCA showing the correlation between different endophytic fungi and their enzyme production abilities. In PCA, a full cross-validation was used to validate the enzyme production ability among different species.</p

UPLC-MS/MS of IAA.

<p>Chromatogram of the culture filtrate of BSS6 (<i>Aureobasidium</i> sp.) showing the correlation between the IAA peak and the standard IAA peak in the same fungal growth media and in water.</p