Characterization and Quantification of the Major Bioactive Compounds in Mexican Purple Tomatoes.

The present work carries out a quantitative analysis of the major bioactive compounds found in the native Mexican purple tomatoes. Total phenolic content ranged from 7.54 to 57.79 mg TPC/g DM, total flavonoid content ranged from 1.89 to 16.93 mg TFC/g DM, total anthocyanin content ranged from 0.29 to 2.56 mg TAC/g DM, and total carotenoid content ranged from 0.11 to 0.75 mg TCC/ g DM. In addition, 14 phenolic acids were identified, among which caffeoylquinic acid derivatives were the most abundant compounds with chlorogenic acid concentration up to 9.680 mg/g DM, together with flavonoids, such as rutin and quercetin-hexoxide. The qualitative analysis also showed the presence of 9 acylated anthocyanins and 2 carotenoids with significant functional features. As for anthocyanins, their chemical structures disclosed special structural features: glycosylated anthocyanins exhibited cis-trans hydroxycinnamic moieties and petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside was reported to be the main anthocyanin, whitin the range of concentrations between 0.160 and 1.143 mg/g DM.

Chemical characterization of yellow-orange and purple varieties of Opuntia ficus-indica fruits and thermal stability of their betalains.

Betalains are plant pigments with biological properties and can be used instead of synthetic colorants to confer color and functional properties to foods. The objective of this work was to carry out the chemical characterization of two varieties of prickly pear of Opuntia ficus-indica, one of yellow-orange coloration (Mandarina) and the other of purple coloration (Vigor), through measurements of chemical parameters and color in pulp, antioxidant activity, total phenolic compounds, and betalain content. Considering the thermolability of betalains and their potential applications in food, the thermal stability and activation energy of betacyanins from Vigor variety and betaxanthins from the Mandarina variety were also evaluated and compared with those from beetroot, the main source of betalains. Results for chemical characterization agreed with previous prickly pear reports of other regions, while the thermal degradation kinetics of betalains showed a first-order degradation pattern with respect to time and temperature treatment. Betacyanins from Vigor prickly pear showed similar thermal stability to those from beetroot, which was reflected in similar values of activation energy, while betaxanthins from Mandarina prickly pear showed a higher stability, and therefore a higher activation energy, than those from beetroot. Based on the results, the prickly pear varieties used in this study can be considered as a good source of betalains with potential applications in food and, in addition, the methodology for the evaluation of thermostability can be used to compare the stability of betalains from different sources in a temperature range of 50-90°C. PRACTICAL APPLICATION: The varieties of prickly pear used in this study can be considered a good source of red-purple and yellow-orange easily extractable pigments. In addition, we report a methodology that can be used for the evaluation of the thermal stability of these pigments and to compare this stability between different plant sources. Gaining knowledge on betalain thermal stability will make it possible to propose specific applications, for example, in processed foods requiring different pigment stabilities.

Extraction of Lipophilic Antioxidants from Native Tomato Using Green Technologies.

Research background: Tomato (Solanum lycopersicum L.) fruit is highly consumed worldwide and contains high amounts of carotenoids and tocopherols, two powerful antioxidants. Native tomato genotypes are rarely used in large-scale market but serve as a reservoir to diversify the species gene pool and can be employed to obtain functional compounds. Extraction methods are currently changing towards cleaner procedures that are more efficient and environmentally friendly, including avoiding toxic or polluting solvents. Experimental approach: In this study, factorial and fractional factorial designs were used to evaluate the efficiency of digestive enzymes, sonication and green solvents to obtain lipophilic antioxidant extracts from native tomato. To monitor the efficiency of the extraction process, spectrophotometric quantification of total carotenoids and antioxidant activity was carried out, and then individual quantification of carotenoids and tocopherols in the extracts was done by HPLC. Results and conclusions: Digestive enzymes and sonication increased the carotenoid content and the antioxidant activity of the obtained extracts when applied individually. However, when these treatments were applied together and in combination with isopropyl acetate, a green solvent, the obtained extracts had the highest carotenoid and tocopherol contents as well as the maximal antioxidant activity. A correlation analysis suggested that antioxidant activity resulted from synergistic effects rather than individual compounds. Tomato extracts were obtained through a rapid and environmentally friendly extraction method and their antioxidant activity was enhanced. Novelty and scientific contribution: Tomato fruits have been the subject of numerous studies; however, functional compound extraction through environmentally friendly methods remains an attractive use of native tomato fruit, enhancing its limited production and harnessing a large amount of tomato product industry. There are few reports where environmentally friendly extraction methods are combined; even rarer are those where green solvents are also used. In this work, the combination of different environmentally friendly extraction methods improved the extraction of carotenoids and tocopherols and allowed to establish a more efficient process. These results could stimulate the use of clean technologies and make the native tomato more attractive for industrial or compound extraction processes.

Characterization of polyphenol oxidase from purple sweet potato (Ipomoea batatas L. Lam) and its affinity towards acylated anthocyanins and caffeoylquinic acid derivatives.

Biochemical characterization of polyphenol oxidase (PPO) present in purple sweet potato (PSP) is a key step in developing efficient methodologies to control oxidative damage caused by this enzyme to the valuable components of PSP, such as caffeoylquinic acid derivatives and acylated anthocyanins. Thus, this work focused on the assessment of the effects of pH, temperature, and chemical agents on the PPO activity as well as characterization of the PPO substrate specificity towards major phenolic compounds found in PSP. The optimum conditions of enzyme activity were pH 7 and a temperature range of 20-30 °C at which phenolic substrates were oxidized with 72.5-99.8% yield. Zn2+ ions remarkably reduced PPO activity while Cu2+ ions improved enzyme performance. The highest substrate preference was shown for 3,4,5-tri-caffeoylquinic and 3,5-di-caffeoylquinic acid, followed by 5-caffeoylquinic and caffeic acid, 3,4- and 4,5-di-caffeoylquinic acids, peonidin-3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside. The highest Km values were found for 4,5-feruloyl-caffeoylquinic acid and catechol.

Caffeoylquinic Acid Derivatives of Purple Sweet Potato as Modulators of Mitochondrial Function in Mouse Primary Hepatocytes.

Owing to their antioxidant properties, caffeoylquinic acid (CQA)-derivatives could potentially improve the impaired metabolism in hepatic cells, however, their effect on mitochondrial function has not been demonstrated yet. Here, we evaluated the impact of three CQA-derivatives extracted from purple sweet potato, namely 5-CQA, 3,4- and 4,5-diCQA, on mitochondrial activity in primary hepatocytes using an extracellular flux analyzer. Notably, an increase of maximal respiration and spare respiratory capacity were observed when 5-CQA and 3,4-diCQA were added to the system indicating the improved mitochondrial function. Moreover, 3,4-diCQA was shown to considerably increase glycolytic reserve which is a measure of cell capability to respond to an energy demand through glycolysis. Conversely, 4,5-diCQA did not modify mitochondrial activity but increased glycolysis at low concentration in primary hepatocytes. All compounds tested improved cellular capacity to oxidize fatty acids. Overall, our results demonstrated the potential of test CQA-derivatives to modify mitochondrial function in hepatic cells. It is especially relevant in case of dysfunctional mitochondria in hepatocytes linked to hepatic steatosis during obesity, diabetes, and metabolic syndrome.

Antimutagenic, Antiproliferative and Antioxidant Properties of Sea Grape Leaf Extract Fractions (Coccoloba uvifera L.).

BACKGROUND: Cancer is a disease characterized by the invasion and uncontrolled growth of cells. One of the best ways to minimize the harmful effects of mutagens is through the use of natural antimutagens. In this regard, the search for new antimutagens that act in the chemoprevention could represent a promising field in this area. OBJECTIVE: In this study biological potential of 11 fractions from Coccoloba uvifera L. leaf hexane extract was evaluated by several in vitro tests. METHODS: Leaves were lyophilized and hexane extraction was performed. The extract was fractionated by column chromatography with hexane, ethyl acetate, and methanol. The antimutagenic (Ames test), antiproliferative (MTT test), and antioxidant capacity (DPPH, ABTS, and ferrous ion chelation) of the fractions were evaluated. RESULTS: Fractions 4, 6, 8, and 9 have antimutagenic activity (against sodium azide in strain TA100), fraction 11 showed antiproliferative capacity (IC50 of 24 ± 9 µg/mL in cells of HCT 116). The fractions with the highest activity were analyzed by HPLC-MS and lupeol, acacetin, and ß-sitosterol were identified. CONCLUSION: This study demonstrates, for the first time, the bioactivity of C. uvifera leaf as a new source of High Biological Value Compounds (HBVC), which can be of interest to the food and pharmaceutical industries.

New acylated cyanidin glycosides extracted from underutilized potential sources: Enzymatic synthesis, antioxidant activity and thermostability.

Interest in anthocyanins has increased remarkably in recent decades, although their wider application has been hampered by instability problems. Thus, this study aimed at developing a strategy to gain access to more stable anthocyanins via enzymatic esterification. For that purpose, three cyanidin derivatives were obtained from underutilized, but easily accessible sources, and their total anthocyanin content was quantified. The purity of cyanidins obtained ranged from 40% to 88% depending on their source. Subsequently, the critical enzymatic reaction conditions were established, and the best results were found using tert-butanol as a solvent, 20 g/L of lipase B from Candida Antarctica, and vinyl cinnamate as acyl donor at ratio 250:1 (acyl donor to anthocyanin). Finally, five new acylated anthocyanin derivatives were synthesized with improved antioxidant activity and thermostability, in comparison to the cyanidin-3-glucoside, which is an advantageous feature for industrial applications.

Quantitative Analysis of the Biologically Active Compounds Present in Leaves of Mexican Sweet Potato Accessions: Phenols, Flavonoids, Anthocyanins, 3,4,5-Tri-Caffeoylquinic Acid and 4-Feruloyl-5-Caffeoylquinic Acid.

Sweet potato is one of the oldest crops cultivated in Mexico, and Mesoamerica is considered as a region with the greatest diversity of this species. Therefore, the present study focused on the evaluation of biologically active compounds, such as caffeoylquinic acid derivatives and flavonoid compounds, in sweet potato leaves of 200 accessions of the main producing regions of Mexico. The analysis of total phenol content (TPC) showed a great variability of concentrations among the examined accessions (54.41 to 284.64 mgTPC/g DW). Likewise, total flavonoid content (TFC) was determined and ranged from 10.01 to 40.17 mgTFC /g DW. Finally, total anthocyanin content (TAC) was evaluated and concentrations obtained varied from 0.05 to 0.98 mgTAC/g DW. Additionally, HPLC analysis of all 200 accessions demonstrated the presence of caffeic acid (CA), 5-caffeoylquinic acid (5-CQA), three isomers of di-caffeoylquinic acid (di-CCA) and 4-feruloyl-5-caffeoylquinic acid (4F-5CQA) in all test samples. Only 21 accessions tested showed the quantitative amount of 3,4,5-tri-caffeoylquinic acid (3,4,5-tri-CQA) with concentrations ranging from 44.73 to 193.22 mg/100 g DW and high content of 4F-5CQA (139.46 to 419.99 mg/100 g DW). The gathered data indicate that leaves of Mexican sweet potatoes are a promising source of phenolic compounds with remarkable nutraceutical potential.

Extraction and Identification of Anthocyanins in Corn Cob and Corn Husk from Cacahuacintle Maize.

Pigmented maize has been extensively studied due to its high anthocyanin content. This study has been focused mainly on kernel, although the whole plant of purple corn is a potential source of anthocyanins. First, general parameters of extraction (solvent system, solvent-to-solid ratio, number of extractions, and acid type) were established depending on the total anthocyanins content. Then, three extraction methods to access anthocyanins were compared: maceration extraction (ME), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE). Since the residual material still possessed an intense color, a further treatment was performed by application of enzymatic-assisted extraction (EAE). Three enzymatic cocktails (Xylanases, Celluclast, and Depol), pH, and temperature were evaluated to establish optimal reaction conditions. Subsequent analysis and identification of the anthocyanins obtained by four different extraction techniques were performed using HPLC and HPLC-mass spectrometry, respectively. The most efficient method was UAE using 20 min of ultrasound (100 W) preceded by sample treatment in the following conditions: ethanol/water/lactic acid mixture (80:19:1), two extractions, 1:10 solvent-to-solid ratio. As a result, anthocyanins from corn cob and corn husk were extracted at concentrations of 24.32 and 25.80 mg/gDW, respectively. No difference in the anthocyanins profile for samples extracted by three different methods was observed. However, an enhanced presence of cyanidin-3-(6''malonyl)glucoside was detected in the sample corresponding to the EAE method. Therefore, the Cahuacintle corn husk can be considered as a competitive source of anthocyanins with the available commercial sources. PRACTICAL APPLICATION: The by-products obtained from Cacahuacintle purple corn can be potentially used as natural colorants thanks to their anthocyanins content. In this work, we established the most efficient extraction method of anthocyanins from corn husk and corn cob, and demonstrated that their anthocyanins profile is comparable to other Peruvian purple corns, which are currently used as natural colorants. Therefore, the extraction procedure described in this study might be scaled-up in an industrial process to get access to anthocyanins from undervalued wastes.

Waste residues from Opuntia ficus indica for peroxidase-mediated preparation of phenolic dimeric compounds.

A methodology to detect peroxidase activity in Opuntia ficus indica cladodes waste extracts was performed and then used towards phenolic compounds. The extracts were able to dimerize three different molecules. Dimeric compounds were produced with yields ranging from 11% to 55%. The influence of H2O2 concentration was also tested, finding better yields when the peroxide-to-substrate ratio was 1:1. Some water-miscible solvents were used trying to increase overall yields, but no-significant positive results were found. In fact, one of them, THF, seemed to inhibit dimerization reaction. Hence, we have tested an alternative natural peroxidase source obtained from the wastes of a local highly-consumed vegetable and studied their enzymatic activity towards the preparation of biologically active, valuable compounds.

Lactic Acid Fermentation of Arabinoxylan From Nejayote by Streptococcus infantarius ssp. infantarius 25124 Isolated From Pozol.

Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) is a lactic acid bacterium (LAB) isolated from pozol, a refreshing beverage prepared by suspending fermented nixtamal (a thermal and alkali-treated maize dough) in water. Although Lactobacillus are the predominant strains in fermented doughs, such as sourdoughs, and non-nixtamalized fermented maize foods, the pozol microbiota is markedly different. This may be the result of the nixtamalization process, which could act as a selective force of some strains. Sii-25124 has been reported as the main amylolytic LAB in pozol; starch is the primary carbon source on nixtamal since monosaccharides and disaccharides are lost during nixtamalization; however, non-amylolytic LAB counts are higher than amylolytic LAB in pozol after 24-h fermentation suggesting that another carbon source is being used by the former bacteria. Hemicellulose (arabinoxylan in maize) becomes available via nixtamalization and is subsequently metabolized by LAB. The aim of this work was to determine whether this bacterium is able to use arabinoxylan as the only carbon source in a defined medium containing arabinoxylan extracted from either nejayote (wash water produced during nixtamal preparation), or beechwood xylan. Xylanase activity in the presence of nejayote arabinoxylan (135.8 ± 48.7 IU/mg protein) was higher than that of beechwood (62.5 ± 19.8 IU/mg protein). Other enzymatic activities, such as arabinofuranosidase and acetyl esterase, were also detected, suggesting the adaptation of the bacterium studied to nixtamal dough. It was concluded that Streptococcus infantarius 25124 isolated from pozol was able to use arabinoxylans, which are present in nixtamal dough, so fermentation does not depend exclusively on free sugars and starch.

ANCUT2, a Thermo-alkaline Cutinase from Aspergillus nidulans and Its Potential Applications.

Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.

Effect of postharvest UV-C treatment on the bacterial diversity of Ataulfo mangoes by PCR-DGGE, survival of E. coli and antimicrobial activity.

Since Mexico is the second largest exporter of mangoes, its safety assurance is essential. Research in microbial ecology and knowledge of complex interactions among microbes must be better understood to achieve maximal control of pathogens. Therefore, we investigated the effect of UV-C treatments on bacterial diversity of the Ataulfo mangoes surface using PCR-DGGE analysis of variable region V3 of 16S rRNA genes, and the survival of E. coli, by plate counting. The UV-C irradiation reduced the microbial load on the surface of mangoes immediately after treatment and the structure of bacterial communities was modified during storage. We identified the key members of the bacterial communities on the surface of fruits, predominating Enterobacter genus. Genera as Lactococcus and Pantoea were only detected on the surface of non-treated (control) mangoes. This could indicate that these genera were affected by the UV-C treatment. On the other hand, the treatment did not have a significant effect on survival of E. coli. However, genera that have been recognized as antagonists against foodborne pathogens were identified in the bands patterns. Also, phenolic compounds were determined by HPLC and antimicrobial activity was assayed according to the agar diffusion method. The main phenolic compounds were chlorogenic, gallic, and caffeic acids. Mango peel methanol extracts (UV-C treated and control mangoes) showed antimicrobial activity against strains previously isolated from mango, detecting significant differences (P < 0.05) among treated and control mangoes after 4 and 12 days of storage. Ps. fluorescens and Ps. stutszeri were the most sensitive.

Immobilization and Biochemical Properties of the Enantioselective Recombinant NStcI Esterase of Aspergillus nidulans.

The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1 h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72 h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, a w of 0.07, and 120 h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures.

Growth inhibition of Streptococcus from the oral cavity by α-amyrin esters.

Five terpenoids were tested by the macrodilution broth method to determine their inhibition activity on cariogenic bacterial growth. In general, α-, β-amyrin and α-amyrin phenylacetate proved to be active, reducing the bacterial viability to less than 20%.

ANCUT2, an extracellular cutinase from Aspergillus nidulans induced by olive oil.

Cutinases are versatile carboxylic ester hydrolases with great potential in many biocatalytic processes, including biodiesel production. Genome sequence analysis of the model organism Aspergillus nidulans reveals four genes encoding putative cutinases. In this work, we purified and identified for the first time a cutinase (ANCUT2) produced by A. nidulans. ANCUT2 is a 29-kDa protein which consists of 255 amino acid residues. Comparison of the amino acid sequence of ANCUT2 with other microbial cutinase sequences revealed a high degree of homology with other fungal cutinases as well as new features, which include a serine-rich region and conserved cysteines. Cutinase production with different lipidic and carbon sources was also explored. Enzyme activity was induced by olive oil and some triacylglycerides and fatty acids, whereas it was repressed by glucose (1%) and other sugars. In some conditions, a 22-kDa post-translational processing product was also detected. The cutinase nature of the enzyme was confirmed after degradation of apple cutin.

Synthesis of chiral alpha-hydroxy amides by two sequential enzymatic catalyzed reactions.

Enantiomerically pure alpha-hydroxy amides have been prepared from the corresponding alpha-oxo esters by the use of a double sequence reaction involving in a first step the highly enantioselective Saccharomyces cerevisiae bioreduction and then in a second step, the resulting alpha-hydroxy esters followed a non-enantiospecific lipase catalyzed aminolysis with n-butylamine reaction. In the first non-organic solvent process, the moistened baker's yeast reduced seven alpha-oxo esters with high conversions degree (93% for one substrate and >99% for the others) and high enantioselectivities [>99% for all the substrates except for ketopantoyl lactone, which gave 88% of enantiomeric excess (ee)]. At the same way, the isolated resulting chiral alpha-hydroxy esters were subjected to the second Candida antarctica lipase fraction B (CAL-B) catalyzed aminolysis in dioxane conducting to the corresponding chiral alpha-hydroxy amides with high conversions degree, between 88 and 99%. Both processes were carried out at 28-30 degrees C.