Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6 U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39 U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70 kDa in C. cladosporioides and ~ 21, 63 and 100 kDa in P. funiculosum. The best results for saccharification (10.11 g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36 h of the hydrolytic process at pH 5 and 50 °C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

Phenolic composition of tomato varieties and an industrial tomato by-product: free, conjugated and bound phenolics and antioxidant activity.

The aim of this study was to isolate, identify and quantify soluble free phenolics, conjugated acid-hydrolysable phenolics (AHP) and alkaline-hydrolysable phenolics, and bound phenolics (BP) fractions from two tomato varieties (saladette and grape) and an industrial tomato by-product, as well as, to determine their antioxidant capacity. Phenolic composition was determined using Folin-Ciocalteu's method and HPLC-DAD. AHP were predominant in grape and saladette tomato extracts (91.47 ± 17.28 mg gallic acid equivalents (GAE) per g dry extract (DE) and 57.41 ± 8.80 mg GAE per g DE, respectively), while BP form was predominant in tomato by-product (51.30 ± 10.91 GAE per g DE). AHP extract of grape tomato presented the highest antioxidant capacity by DPPH assay (252.35 ± 42.55 µmol trolox equiv (TE) per g DE). In the case of ORAC assay, AHP fractions from both grape (1005.19 ± 138.52 µmol TE per g DE) and saladette tomatoes (804.16 ± 131.45 µmol TE per g DE), and BP fraction from by-product (852.40 ± 71.46 µmol TE per g DE) showed the highest ORAC values. Caffeic acid was the most abundant phenolic acid and it was found mainly in its conjugated forms. Naringenin was the most abundant flavonoid and it was mainly detected in bound form. Our analysis allowed a better characterization of phenolic compounds in whole tomato and by-product, remarking the importance of the fractionation. The valorization of the industrial tomato by-product, through the use of its different fractions of phenolic antioxidant compounds, could generate additional income to the tomato industry and reduce the waste disposal problem.

Bioactive compounds and antioxidant activity in scalded Jalapeño pepper industrial byproduct (Capsicum annuum).

Bioactive compounds and antioxidant activity were evaluated from industrial Jalapeño pepper byproducts and simulated non processed byproducts from two Mexican states (Chihuahua and Sinaloa) to determine their value added potential as commercial food ingredients. Aqueous 80% ethanol produced about 13% of dry extract of polar compounds. Total phenolic content increased and capsaicin and dihydrocapsaicin decreased on scalding samples (80 °C, 2 min) without affecting ascorbic acid. The major phenolic compounds, rutin, epicatechin and catechin comprised 90% of the total compounds detected by HPLC of each Jalapeño pepper byproducts. ORAC analysis showed that the origin and scalding process affected the antioxidant activity which correlated strongly with capsaicin content. Although scalding decreased capsaicinoids (up to 42%), phenolic content by (up to 16%), and the antioxidant activity (variable). Jalapeño pepper byproduct is a good source of compounds with antioxidant activity, and still an attractive ingredient to develop useful innovative products with potential food/non-food applications simultaneously reducing food loss and waste.

Phenolic profile and antioxidant activity from non-toxic Mexican Jatropha curcas L. shell methanolic extracts.

Jatropha curcas seed shells are the by-product obtained during oil extraction process. Recently, its chemical composition has gained attention since its potential applications. The aim of this study was to identify phenolic compounds profile from a non-toxic J. curcas shell from Mexico, besides, evaluate J. curcas shell methanolic extract (JcSME) antioxidant activity. Free, conjugate and bound phenolics were fractionated and quantified (606.7, 193.32 and 909.59 µg/g shell, respectively) and 13 individual phenolic compounds were detected by HPLC. The radical-scavenging activity of JcSME was similar to Trolox and ascorbic acid by DPPH assay while by ABTS assay it was similar to BHT. Effective antioxidant capacity by ORAC was found (426.44 ± 53.39 µmol Trolox equivalents/g shell). The Mexican non-toxic J. curcas shell is rich in phenolic compounds with high antioxidant activity; hence, it could be considerate as a good source of natural antioxidants.

Phenolic content and antioxidant and antimutagenic activities in tomato peel, seeds, and byproducts.

The phenolic content and antioxidant and antimutagenic activities from the peel and seeds of different tomato types (grape, cherry, bola and saladette type), and simulated tomato industrial byproducts, were studied. Methanolic extracts were used to quantify total phenolic content, groups of phenolic compounds, antioxidant activities, and the profile of phenolic compounds (by HPLC-DAD). Antimutagenic activity was determined by Salmonella typhimurium assay. The total phenolic content and antioxidant activity of tomato and tomato byproducts were comparable or superior to those previously reported for whole fruit and tomato pomace. Phenolic compounds with important biological activities, such as caffeic acid, ferulic acid, chlorogenic acids, quercetin-3-ß-O-glycoside, and quercetin, were quantified. Differences in all phenolic determinations due to tomato type and part of the fruit analyzed were observed, peel from grape type showing the best results. Positive antimutagenic results were observed in all samples. All evaluated materials could be used as a source of potential nutraceutical compounds.

PvLOX2 silencing in common bean roots impairs arbuscular mycorrhiza-induced resistance without affecting symbiosis establishment.

The arbuscular mycorrhizal (AM) symbiosis is an intimate association between specific soil-borne fungi and the roots of most land plants. AM colonisation elicits an enhanced defence resistance against pathogens, known as mycorrhizal-induced resistance (MIR). This mechanism locally and systemically sensitises plant tissues to boost their basal defence response. Although a role for oxylipins in MIR has been proposed, it has not yet been experimentally confirmed. In this study, when the common bean (Phaseolus vulgaris L.) lipoxygenase PvLOX2 was silenced in roots of composite plants, leaves of silenced plants lost their capacity to exhibit MIR against the foliar pathogen Sclerotinia sclerotiorum, even though they were colonised normally. PvLOX6, a LOX gene family member, is involved in JA biosynthesis in the common bean. Downregulation of PvLOX2 and PvLOX6 in leaves of PvLOX2 root-silenced plants coincides with the loss of MIR, suggesting that these genes could be involved in the onset and spreading of the mycorrhiza-induced defence response.

Angiotensin-converting enzyme inhibitory and antioxidative activities and functional characterization of protein hydrolysates of hard-to-cook chickpeas.

BACKGROUND: The potential use of hard-to-cook (hardened) chickpeas to obtain value-added functional food ingredients was evaluated. For that purpose, some nutraceutical and functional attributes of several chickpea protein hydrolysates (CPHs) prepared from both fresh and hard-to-cook grains were evaluated. RESULTS: All the CPHs prepared from both fresh and hard-to-cook grains, with the enzymes alcalase, pancreatin and papain, showed high angiotensin converting enzyme inhibitory (ACE-I) activity with IC50 values ranging from 0.101 to 37.33 µg mL⁻¹; similarly, high levels of antioxidant activity (around 18.17-95.61 µmol Trolox equivalent antioxidant capacity µg⁻¹ CPH) were obtained through both the 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) methods. Regarding functional characterization of the CPHs, oil absorption values ranged from 1.91 to 2.20 mL oil g⁻¹ CPH, with water solubility almost 100% from pH 7 to 10. CONCLUSION: The high antioxidant and ACE-I activities as well as the good functional properties of the CPH prepared from both fresh and hard-to-cook grains, suggest its use in food formulations with value added in human health.

Modification of the amaranth 11S globulin storage protein to produce an inhibitory peptide of the angiotensin I converting enzyme, and its expression in Escherichia coli.

Amarantin is the predominant seed storage protein from amaranth. It shows a high content of essential amino acids, making this protein important from a nutritional viewpoint. The protein has two disulfide linked subunits: acidic and basic. Acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. In order to improve its functionality, the primary structure was modified in the third variable region of globulins 11S, by inserting four Val-Tyr antihypertensive peptides in tandem. The designed plasmid was expressed in Escherichia coli Origami (DE3) and then the expressed protein was purified. Mass spectrometry analysis was used to corroborate the identity of the protein by peptide mass fingerprinting; also, the modified peptide was fragmented and sequenced by mass spectrometry, corroborating thus the inserted residues. The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50) 0.064 mg ml(-1)); it was nearly eightfold more active than the nonmodified protein. In spite that the nonmodified subunit is less active, its activity is comparable with other hydrolyzed proteins reported as high active inhibitors. The expressed and purified subunit after its engineered modification, may be useful for preventing hypertension and for other medical purposes.

Expression and characterization of the acidic subunit from 11S Amaranth seed protein.

Amarantin acidic subunit has the potential to be employed as a functional and a nutraceutical protein. To evaluate both possibilities this protein was produced in recombinant Escherichia coli Origami (DE3) harboring the expression plasmid pET-AC6His. Three different expression factors were assayed: inductor concentration, temperature and time of the amarantin acidic subunit accumulation. The results indicated that a 0.3 mmol/L concentration of isopropyl-beta-D-thiogalactoside, at 37 degrees C and 6 h after induction were favorable for high expression of amarantin acidic subunit, mostly in the form of inclusion bodies. The protein was purified from soluble fraction by immobilized metal affinity chromatography, up to 30 mg amarantin acidic subunit/L Terrific broth culture were obtained. Sucrose density gradient ultracentrifugation analysis of the expressed soluble amarantin acidic subunit revealed that it was assembled in monomers. The expression of the amarantin acidic subunit, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.

Endoplasmic reticulum-retention C-terminal sequence enhances production of an 11S seed globulin from Amaranthus hypochondriacus in Pichia pastoris.

The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.

One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco.

Amarantin, an 11S globulin, is one of the most important storage proteins of amaranth seeds, with relevant nutritional-functional and nutraceutical characteristics. Its cDNA was cloned in-frame with a sequence encoding a polyhistidine tag and expressed under the direction of a 35S promoter in transgenic tobacco seeds. The presence of a (His)(6) tag on the polypeptide permitted a high-yield single-step purification using immobilized metal-ion affinity chromatography and rapid characterization. Purified His-tag amarantin accounted for up to 5% of total soluble seed protein. Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight (53 kDa) and was correctly processed into an acidic polypeptide (32 kDa) with isoelectric point (pI) of 5.58 and a basic polypeptide (21 kDa) with pI of 9.24, linked by a disulfide bridge. Moreover, His-tag amarantin was assembled into both homo- and hetero-hexameric 11S structures. These results show that the His tag did not change the biochemical and physicochemical properties of amarantin. The strategy presented here for rapid and high-yield expression and purification procedure should facilitate structure-function studies for this nutritional protein.

Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli.

DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.

Safety assessment by in vitro digestibility and allergenicity of genetically modified maize with an amaranth 11S globulin.

Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult to perform. Thus, the objective of this work was to assess the food safety of genetically modified maize with an amaranth globulin protein termed amarantin. Transgenic maize lines evaluated showed, in relation to nontransgenic, 4-35% more protein and 0-44% higher contents of specific essential amino acids. Individual sequence analysis with known amino acid sequences, reported as allergens, showed that none of these IgE elicitors were identified in amarantin. Amarantin was digested within the first 15 min by Simulated Gastric Fluid treatment as observed by Western blot. Expressed amarantin did not induce important levels of specific IgE antibodies in BALB/c mice, as analyzed by ELISA. We conclude that the transgenic maize with amarantin is not an important allergenicity inducer, just as nontransgenic maize.