Publicaciones 2012

Shene, C., Canquil, N., Jorquera, M., Pinelo, M., Rubilar, M., Acevedo, F., Vergara, C., von Baer, D., Mardones, C. 2012. In vitro activity on human gut bacteria of Murta leaf extracts (Ugni molinae Turcz.), a native plant from Southern Chile. Journal of Food Science. 77; 6;, M323-M329.


Despite the fact that murta infusions have been used to treat gut/urinary infections by native Chileans for centuries, the mechanisms promoting such effects still remain unclear. As a first attempt to unravel these mechanisms, human fecal samples were incubated in a medium containing water extract of murta leaves (ML) and the growth of different bacterial groups was evaluated. Control incubations were made in media containing fructooligosaccharides (FOS) and glucose as a carbon source. Phenolic compounds in the ML extract, likely promoters of bioactivity, were identified by HPLC-DAD-MS(n) . Concentrations (log₁₀ CFU/mL) of bifidobacteria and lactobacilli in media containing the extract and FOS were 7.33 ± 0.05/4.95 ± 0.20 and 6.44 ± 0.22/6.05 ± 0.06, respectively. Clostridia, anaerobes and Enterobacteriaceae grew to a similar extent in media containing murta extract and FOS. In vitro tests (disk diffusion) showed that Gram-positive (Bacillus and Paenibacillaceae) and Gram-negative (Enterobacteriaceae) bacteria isolated from fecal samples were sensitive to both water and 50/50 ethanol/water extracts of ML (28.4 μg gallic acid equivalents). At this concentration, the antimicrobial activity of ML extracts was significantly (P < 0.05) lower than that of penicillin (10 U), whereas the difference between activity of ML extracts and gentamicine (10 μg) was no significant (P > 0.05). No evidence of dependency between the antimicrobial activity of ML extracts and the enzymatic capability of the sensitive strains was found.

Pablo D. Cárdenas, Humberto A. Gajardo, Terry Huebert, Isobel A. Parkin, Federico L. Iniguez-Luy and María L. Federico. (2012).  Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae. Theoretical and Applied Genetics. 124; 7; 1215-1228.


The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSYhomologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSYgenes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.


Flax seed mucilage (SM) presents specific biological activities useful for the food and pharmaceutical industries. Understanding the population structure, genetic diversity and linkage disequilibrium (LD) of germplasm varying for mucilage content is pivotal for the identification of genes and quantitative trait loci underlying mucilage variation by association mapping (AM). In this study, 150 microsatellite loci were used to assess the population structure, genetic diversity and LD of a set of 60 flax cultivars/accessions capturing the breadth of SM variation in flax germplasm. STRUCTURE analysis and similarity-based methods revealed the presence of three populations reflecting mainly their geographic origins (South Asia, South America and North America), and the impact of germplasm exchange within and between North American flax breeding programs. Analysis of molecular variance showed that 78.32% of the genetic variation resided within populations and 21.68% among populations. The phi-statistic (Φst) value of 0.22 confirmed the presence of a strong population structure. A total of 408 alleles were detected, with the South American population capturing the highest overall diversity. However, the genetic diversity was narrow, as indicated by the small number of alleles per locus (2.72) and gene diversity (mean = 0.34). LD was significant between 3.9% (r 2) and 36.2% (D′) of the loci pairs (FDR < 0.05). The mean r 2 and D′ were 0.26 and 0.53, respectively. The results suggest that the collection could be useful in AM studies aimed at the discovery of genes/alleles involved in SM; however a greater diversity may be required to improve the AM resolution.

Snowdon, R. J. and Iniguez Luy, . F. L. (2012). Potential to improve oilseed rape and canola breeding in the genomics eraPlant Breeding. 131; 3; 351-360.


High-throughput genomics technologies today offer unprecedented possibilities for gene discovery, complex trait analysis by genome-wide association studies, global gene expression analyses, genomic selection and predictive breeding strategies. Dissection of the complex Brassica napus genome using mapping-by-sequencing techniques provides a powerful bridge between genetic maps and genome sequences. The completed sequence of the Brassica rapa A genome and the expected forthcoming publication of the C genome (Brassica oleracea) will greatly accelerate the release of public reference sequences for B. napus (genome AC). Dramatically falling DNA costs for targeted or genomic resequencing and the availability of a new, high-density B. napus single-nucleotide polymorphism (SNP) array open the way for considerably more efficient mining and exploitation of genetic variation within the primary and secondary gene pools of B. napus. In this review, we outline some of the most significant recent advances in high-throughput genomics of Brassica crops and their potential impact on germplasm development and breeding of oilseed rape and canola in the coming years and decades.

Curilem Saldías M, Villarroel Sassarini F, Muñoz Poblete C, Vargas Vásquez A, Maureira Butler I. (2012). Image Correlation Method for DNA Sequence AlignmentPLoS ONE. 7; 6; e39221.


The complexity of searches and the volume of genomic data make sequence alignment one of bioinformatics most active research areas. New alignment approaches have incorporated digital signal processing techniques. Among these, correlation methods are highly sensitive. This paper proposes a novel sequence alignment method based on 2-dimensional images, where each nucleic acid base is represented as a fixed gray intensity pixel. Query and known database sequences are coded to their pixel representation and sequence alignment is handled as object recognition in a scene problem. Query and database become object and scene, respectively. An image correlation process is carried out in order to search for the best match between them. Given that this procedure can be implemented in an optical correlator, the correlation could eventually be accomplished at light speed. This paper shows an initial research stage where results were “digitally” obtained by simulating an optical correlation of DNA sequences represented as images. A total of 303 queries (variable lengths from 50 to 4500 base pairs) and 100 scenes represented by 100 x 100 images each (in total, one million base pair database) were considered for the image correlation analysis. The results showed that correlations reached very high sensitivity (99.01%), specificity (98.99%) and outperformed BLAST when mutation numbers increased. However, digital correlation processes were hundred times slower than BLAST. We are currently starting an initiative to evaluate the correlation speed process of a real experimental optical correlator. By doing this, we expect to fully exploit optical correlation light properties. As the optical correlator works jointly with the computer, digital algorithms should also be optimized. The results presented in this paper are encouraging and support the study of image correlation methods on sequence alignment.

Parra-Gonzalez Lorena, Aravena-Abarzua Gabriela, Navarro-Navarro Cristell, Udall Joshua, Maugham Peter, Peterson Louis, Salvo-Garrido Haroldo, Maureira-Butler Iván. (2012). Yellow lupin (Lupinus luteus L.) transcriptome sequencing: molecular marker development and comparative studies. BMC Genomics. 13; 425.



Yellow lupin (Lupinus luteus L.) is a minor legume crop characterized by its high seed protein content. Although grown in several temperate countries, its orphan condition has limited the generation of genomic tools to aid breeding efforts to improve yield and nutritional quality. In this study, we report the construction of 454-expresed sequence tag (EST) libraries, carried out comparative studies between L. luteus and model legume species, developed a comprehensive set of EST-simple sequence repeat (SSR) markers, and validated their utility on diversity studies and transferability to related species.


Two runs of 454 pyrosequencing yielded 205 Mb and 530 Mb of sequence data for L1 (young leaves, buds and flowers) and L2 (immature seeds) EST- libraries. A combined assembly (L1L2) yielded 71,655 contigs with an average contig length of 632 nucleotides. L1L2 contigs were clustered into 55,309 isotigs. 38,200 isotigs translated into proteins and 8,741 of them were full length. Around 57% of L. luteus sequences had significant similarity with at least one sequence of Medicago, Lotus, Arabidopsis, or Glycine, and 40.17% showed positive matches with all of these species. L. luteus isotigs were also screened for the presence of SSR sequences. A total of 2,572 isotigs contained at least one EST-SSR, with a frequency of one SSR per 17.75 kbp. Empirical evaluation of the EST-SSR candidate markers resulted in 222 polymorphic EST-SSRs. Two hundred and fifty four (65.7%) and 113 (30%) SSR primer pairs were able to amplify fragments from L. hispanicus and L. mutabilis DNA, respectively. Fifty polymorphic EST-SSRs were used to genotype a sample of 64 L. luteus accessions. Neighbor-joining distance analysis detected the existence of several clusters among L. luteus accessions, strongly suggesting the existence of population subdivisions. However, no clear clustering patterns followed the accession’s origin.


L. luteus deep transcriptome sequencing will facilitate the further development of genomic tools and lupin germplasm. Massive sequencing of cDNA libraries will continue to produce raw materials for gene discovery, identification of polymorphisms (SNPs, EST-SSRs, INDELs, etc.) for marker development, anchoring sequences for genome comparisons and putative gene candidates for QTL detection.

Rubilar M., Morales E., Sáez R., Acevedo F., Palma B., Villarroel M., Shene C. (2012). Polyphenolic fractions improve the oxidative stability of microencapsulated linseed oil. European Journal of Lipid Science and Technology. 114; 7; 760-771.


The main objective of this study was to evaluate the effect of incorporating polyphenolic-enriched fractions from murta leaves on the oxidative stability of linseed oil microencapsulated by spray drying. For this purpose, polyphenol-enriched fractions from murta leaves were separated by gel permeation chromatography and chemically characterized. The oxidative stability of microencapsulated linseed oil (MLO) with antioxidants was evaluated in storage conditions at 25°C for 40 days. The antioxidant effects of the polyphenolic fractions and commercial antioxidants (BHT and trolox) on microencapsulated oil were evaluated by the value of conjugated dienes, peroxide, and p-anisidine. In the initiation step of the oxidation, no significant oxidation delay (p>0.05) in MLO containing fractions F6, F8, or BHT and trolox was observed. However, in the termination step of the oxidation, the addition of fractions F6, F8, and BHT and trolox decreases significantly (p ≤ 0.05) the rancidity in MLO. Furthermore, the results of this study demonstrated the importance of the addition of natural antioxidants such as fractions of murta leaf extract in microencapsulated linseed oil to increase its resistance to oxidation.

Practical applications: For incorporating linseed oil, a source of omega-3 fatty acids, in the diet it is necessary to protect it against oxidative rancidity, the main cause of deterioration that affects food with a high unsaturated fat content. Microencapsulation is effective in retarding or suppressing the oxidation of unsaturated fatty acids and natural plants extracts are effective in inhibiting the lipid oxidation of microencapsulated oil. The use of process technology and a natural additive is expected to increase storage stability and enable its use in dry foods such as instant products. Linseed oil can be used in human nutrition as well as in animal feed as a replacement for fish oil.

Rubilar, M.; Morales, E.; Contreras, K.; Ceballos,C.; Acevedo, F.; Villarroel, M.; Shene, C. (2012). Development of a soup powder enriched with microencapsulated linseed oil as a source of omega-3. European Journal of Lipid Science and Technology. 114; 4; 423-433.


The main objective of this study was to develop an optimized formulation of soup powder enriched with omega-3 fatty acids. For this purpose, the process conditions for optimizing the microencapsulation efficiency (ME) of linseed oil by spray drying were determined using the Taguchi methodology with an orthogonal array L4(23). The effect of the variables on the ME, such as wall material concentration (25 and 30%), linseed oil concentration (14 and 20%), and wall material type (gum arabic GA; and a mixture maltodextrin/GA), was evaluated. The oxidative stability of the microcapsules obtained were determined by the Rancimat method, and a morphological and size characterization of microcapsules was performed by scanning electronic microscopy, confocal microscopy, and laser diffraction. The optimization of the soup formulation was reached by means of RSM, using the central composite design, two control factors (salty taste and consistency), and 11 design points. The hedonic test was applied to measure the acceptability of the optimized formulation. Chemical characterization of optimized soup and its oxidative stability were also evaluated. This study resulted in a healthy soup enriched with omega-3 which was highly acceptable to consumers.

Practical applications: Linseed oil is a healthy and nutritive oil, very rich in unsaturated fatty acids such as omega-3. In addition to protecting the oil against oxidative damage, the microencapsulation of oils in a polymeric matrix (mainly polysaccharides and proteins) also offers the possibility of controlled release of the lipophilic functional ingredient and can be useful for the supplementation of foods with PUFA. The soup powder enriched with microencapsulated linseed oil as a source of omega-3 formulated in this study will contribute to the development of foods according to the functionality requirements of current consumers and markets.

Uquiche E., Huerta E., Sandoval A., del Valle J. (2012) Effect of boldo (peumus boldus M.) pretreatment on kinetics of supercritical CO2 extraction of essencial oilJournal of Food Engineering, 109, 2, 230-237.

This work examined the effect of the solid matrix on supercritical carbon dioxide (SC CO2) extraction of essentials oils from boldo leaves (Peumus boldus M.) subjected to rapid decompression of a CO2-impregnated sample, conventional milling, and low-temperature milling. Low-temperature conditioning prior to milling decreased heat-driven losses of volatile compounds during milling, as attested by a higher extract yield for low-temperature (10.8 g extract/kg extract-free substrate) than conventionally (9.63 g/kg) milled sample. Extract yield was even larger for the rapidly decompressed sample (11.4 g/kg). Results of SC CO2 extraction experiments carried out at 40 °C and 10 MPa were adjusted to a diffusional model using the effective diffusivity of the extract in the solid matrix (De) and a single partition of essential oils between solid substrate and SC CO2 as best-fitting parameters. A microstructural factor (FM), which was estimated as the ratio between the binary diffusion coefficient of the essential oil in SC-CO2 under extraction conditions and De, was used to characterize the effect of sample pretreatment on extraction rates. Values of FM for rapidly decompressed (202) and low-temperature milled (1740) samples were smaller than value for conventionally milled samples (2200), which revealed that the two first treatments disrupted secretory cavities in boldo leaves more effectively than the third. This was confirmed by light microscopy observations. The work included also measurements using oregano bracts (Origanum vulgare L.) as the substrate to confirm literature reports on the SC CO2 extraction of pretreated bracts and to serve as a reference to our main results with boldo leaves. Trends with oregano coincided with those of boldo.

Uquiche, E., Fica, X., Salazar, K., del Valle, J.M. (2012). Time fractionation of minor lipids from cold-pressed rapeseed cake using supercritical CO2. Journal of the American Oil Chemists’ Society. 89; 6; 1135-1144.


This work explored the possibility of using supercritical carbon dioxide (SC-CO2) to achieve fractionation of pre-pressed rapeseed (Brassica napus) cake oil at 30–50 MPa, at 40 or 80 °C, and increase the concentration of minor lipids (sterols, tocopherols, carotenoids) in the oil. Minor lipids are partially responsible for desirable antioxidant effects that protect against degradation and impart functional value to the oil. The weight and concentration of minor lipids in oil fractions collected during the first 60 min were analyzed. Cumulative oil yield increased with pressure, and with temperature at ≥40 MPa, but was lower at 80 °C than at 40 °C when working at pressure ≤35 MPa. Differences in solubility between the oil and minor lipids explained fractionation effects that were small for tocopherols. Unlike tocopherols, which are more soluble in SC-CO2 than the oil, sterols and carotenoids are less soluble than the oil, and their concentration increased in the later stages of extraction, particularly at ≥40 MPa, when there was not enough oil to saturate the CO2 phase. Because of the fractionating effects on rapeseed oil composition, there was an increase in the antioxidant activity of the oil in the second half as compared to the first half of the extraction. Consequently, this study suggests that SC-CO2 extraction could be used to isolate vegetable oil fractions with increased functional value.

Acevedo, Francisca; Rubilar, Mónica; Shene, Carolina; Navarrete, Patricia; Romero, Fernando; Rabert, Claudia; Jolivet, Pascale; Valot, Benoit; Chardot, Thierry. (2012). Seed oil bodies from non sequenced species: Gevuina avellana and Madia sativa.  Journal of Agricultural and Food Chemistry. 60; 28; 6994-7004


In this study, oil bodies (OBs) from Gevuina avellana (OBs-G) and Madia sativa (OBs-M) were isolated and characterized. Microscopic inspection revealed that the monolayer on OB-G was thinner compared to that on OB-M. Cytometric profiles regarding size, complexity, and staining for the two OB sources were similar. Fatty acid to protein mass ratio in both OBs was near 29, indicating high lipid enrichment. OBs-G and OBs-M showed a strong electrostatic repulsion over wide ranges of pH (5.5–9.5) and NaCl concentration (0–150 mM). Proteins displaying highly conserved sequences (steroleosins and aquaporins) in the plant kingdom were identified. The presence of oleosins was immunologically revealed using antibodies raised against Arabidopsis thaliana oleosins. OBs-G and OBs-M exhibited no significant cytotoxicity against the cells. This is the first report about the isolation and characterization of OBs-G and OBs-M, and this knowledge could be used for novel applications of these raw materials.

Rubilar, M., Villarroel, M., Paillacar, M., Moenne-Locóz, D., Shene, C., Biolley, E., Acevedo, F. (2012) Native black Michuñe potato variety: characterization, frying conditions and sensory evaluation. Archivos Latinoamericanos de Nutrición.  62; 1; 87-93.

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Uquiche, E.; Romero, V.; Ortiz, J.  and  Del Valle, J. M.(2012) Extraction of oil and minor lipids from cold-press rapeseed cake with supercritical CO2. Brazilian Journal of Chemical Engineering. 29; 3; 585-598.


This study examines the extraction of oil from cold-press rapeseed cake using Supercritical CO2(SC-CO2). The effects of pressure (20, 30, and 40 MPa), temperature (40, 50, and 60 ºC), and extraction time (60, 90, and 120 min) on oil yield and composition (tocopherols and carotenoids) were studied using response surface design. The results indicated that pressure influenced the most the yield of oil, followed by temperature and extraction time. Extraction time had no effect on oil composition. Extraction pressure and temperature did not affect the tocopherol concentration of the oil to a great extent, whereas temperature had no affect in its carotenoid concentration. A comparison was made between the relative qualities of oil extracted with SC-CO2at 40 MPa and 60 ºC and with n-hexane. Neither solvent affected the unsaponifiable matter content or the composition of phytosterols (mainly β-sitosterol, campesterol and brassicasterol) of the oils, although there was a significant difference (p<0.05) in tocopherol. Extraction with SC-CO2at 40 MPa and 60 ºC is recommended to obtain rapeseed-oil enriched with tocopherols and carotenoids as important functional components.

Soto-Cerda BJ, Cloutier S. (2012). Chapter 3: Association mapping in plant genomes, in Genetic Diversity in Plants, Mahmut Çalişkan, (Ed.), InTech, ISBN 978-953-307-1250-1. pp. 29-55.


J. Sineiro, M. Rubilar, M. Cascante, E. Álvarez , M. Sánchez , M. J. Núñez (2012) Potential Therapeutic Applications of Common Agro-Food Byproducts and Chilean Wild PlantsEmerging Trends in Dietary Components for Preventing and Combating Disease. pp. 117-130


Procyanidins from grapeseed, grape pomace and pine bark were obtained by extraction with ethanol and further solvent partitioning to obtain a fraction soluble in water and both ethyl acetate, or directly recovered from Amberlite XAD16. Then, proanthocyanidin-enriched fractions were obtained by gel filtration through Sephadex LH-20 or Toyopearl HW-40F. Comparison of galloylated and non-galloylated procyanidins showed the influence of galloylation on the induction of apoptosis in colon cancer cells and in melanoma cells. Fractions from grapes showing galloylation percentages between 15 and 34 % and polymerization degrees between 1.7 and 3.4 induced apoptosis. The cardioprotective potential of proanthocyanidin-containing fractions from grape pomace was studied by measuring the inhibition of human endothelial NADPH oxidase. IC50 values for NADPH inhibition were in the range 2.5-3.4 mg/L. Extracts from green parts of murta (Ugni molinae Turcz.) and maqui (Aristotelia chilensis) are rich in flavonols (kaempferol, myricetin, rutin,- among others) and the fruits rich in anthocyanins. Murta and maqui extracts showed anti-hemolytic activity and inhibited alpha-amylase, although not alpha-glucosidase.