Pressurized Hot Water Extraction and Bio-Hydrogels Formulation with Aristotelia chilensis [Mol.] Stuntz Leaves.

Aristotelia chilensis is a plant rich in phenolics and other bioactive compounds. Their leaves are discarded as waste in the maqui berry industry. A new application of these wastes is intended by the recovery of bioactive compounds using pressurized hot water extraction with conventional or microwave heating. Both technologies have been selected for their green character regarding the type of solvent and the high efficiency in shorter operation times. Extractions were performed in the temperature range 140-200 °C with a solid/liquid ratio of 1:15 (w:w). The extracts' total phenolic content, antioxidant capacity, and saccharides content obtained with both heating methods were measured. Additionally, the thermo-rheological properties of the gelling matrix enriched with these extracts were analyzed. Optimum conditions for lyophilized extracts were found with conventional heating, at 140 °C and 20 min extraction; 250.0 mg GAE/g dry extract and 1321.5 mg Trolox/g dry extract. Close to optimum performance was achieved with microwave heating in a fraction of the time (5 min) at 160 °C (extraction), yielding extracts with 231.9 mg GAE/g dry extract of total phenolics and antiradical capacity equivalent to 1176.3 mg Trolox/g dry extract. Slightly higher antioxidant values were identified for spray-dried extracts (between 5% for phenolic content and 2.5% for antioxidant capacity). The extracts obtained with both heating methods at 200 °C contained more than 20% oligosaccharides, primarily glucose. All the formulated gelling matrices enriched with the obtained extracts displayed intermediate gel strength properties. The tested technologies efficiently recovered highly active antioxidant extracts, rich in polyphenolics, and valuable for formulating gelling matrices with potential applicability in foods and other products.

Bioactive Properties of Marine Phenolics.

Phenolic compounds from marine organisms are far less studied than those from terrestrial sources since their structural diversity and variability require powerful analytical tools. However, both their biological relevance and potential properties make them an attractive group deserving increasing scientific interest. The use of efficient extraction and, in some cases, purification techniques can provide novel bioactives useful for food, nutraceutical, cosmeceutical and pharmaceutical applications. The bioactivity of marine phenolics is the consequence of their enzyme inhibitory effect and antimicrobial, antiviral, anticancer, antidiabetic, antioxidant, or anti-inflammatory activities. This review presents a survey of the major types of phenolic compounds found in marine sources, as well as their reputed effect in relation to the occurrence of dietary and lifestyle-related diseases, notably type 2 diabetes mellitus, obesity, metabolic syndrome, cancer and Alzheimer's disease. In addition, the influence of marine phenolics on gut microbiota and other pathologies is also addressed.

Bioactive Polyphenols from Southern Chile Seaweed as Inhibitors of Enzymes for Starch Digestion.

The increment of non-communicable chronic diseases is a constant concern worldwide, with type-2 diabetes mellitus being one of the most common illnesses. A mechanism to avoid diabetes-related hyperglycemia is to reduce food digestion/absorption by using anti-enzymatic (functional) ingredients. This research explored the potential of six common Chilean seaweeds to obtain anti-hyperglycemic polyphenol extracts, based on their capacity to inhibit key enzymes related with starch digestion. Ethanol/water hot pressurized liquid extraction (HPLE), which is an environmentally friendly method, was studied and compared to conventional extraction with acetone. Total polyphenols (TP), antioxidant activity, cytotoxicity and inhibition capacity on α-glucosidase and α-amylase were analyzed. Results showed that the Durvillaea antarctica (cochayuyo) acetone extract had the highest TP content (6.7 ± 0.7 mg gallic acid equivalents (GAE)/g dry seaweed), while its HPLE ethanol/water extract showed the highest antioxidant activity (680.1 ± 11.6 µmol E Trolox/g dry seaweed). No extract affected cell viability significantly. Only cochayuyo produced extracts having relevant anti-enzymatic capacity on both studied enzymes, showing a much stronger inhibition to α-glucosidase (even almost 100% at 1000 µg/mL) than to α-amylase. In conclusion, from the Chilean seaweeds considered in this study, cochayuyo is the most suitable for developing functional ingredients to moderate postprandial glycemic response (starchy foods), since it showed a clear enzymatic inhibition capacity and selectivity.

The Impact of Temperature and Ethanol Concentration on the Global Recovery of Specific Polyphenols in an Integrated HPLE/RP Process on Carménère Pomace Extracts.

Sequential extraction and purification stages are required to obtain extracts rich in specific polyphenols. However, both separation processes are often optimized independently and the effect of the integrated process on the global recovery of polyphenols has not been fully elucidated yet. We assessed the impact of hot-pressurized liquid extraction (HPLE) conditions (temperature: 90-150 °C; ethanol concentration: 15%-50%) on the global recovery of specific phenolic acids, flavanols, flavonols and stilbenes from Carménère grape pomace in an integrated HPLE/resin purification (RP) process. HPLE of phenolic acids, flavanols and stilbenes were favored when temperature and ethanol concentration increased, except for chlorogenic acid which showed an increment of its Gibbs free energy of solvation at higher ethanol contents. Ethanol concentration significantly impacted the global yield of the integrated HPLE/RP process. The lower the ethanol content of the HPLE extracts, the higher the recovery of phenolic acids, flavanols and stilbenes after RP, except for flavonols which present more polar functional groups. The best specific recovery conditions were 150 °C and ethanol concentrations of 15%, 32.5% and 50% for phenolic acids, flavanols and stilbenes, and flavonols, respectively. At 150 °C and 32.5% of ethanol, the extracts presented the highest total polyphenol content and antioxidant capacity. The integrated HPLE/RP process allows a selective separation of specific polyphenols and eliminates the interfering compounds, ensuring the safety of the extracts at all evaluated conditions.

The Antioxidant and Safety Properties of Spent Coffee Ground Extracts Impacted by the Combined Hot Pressurized Liquid Extraction-Resin Purification Process.

Hot pressurized liquid extraction has been used to obtain polyphenols; however, its operating conditions can generate hydroxymethylfurfural, a potential human carcinogen. The addition of ethanol can reduce process temperatures and retain extraction efficiencies, but the ethanol may reduce the recovery of polyphenols in the subsequent purification stage, affecting the antioxidant properties of the extracts. This study evaluates a combined hot pressurized liquid extraction-resin purification process to obtain polyphenol extracts from spent ground coffee reduced in hydroxymethylfurfural. A multifactorial design was developed to determine the combined effect of the extraction (ethanol content: 0-16% and temperature: 60-90 °C) and purification (ethanol: 60-80%) conditions on some chemical properties of the extracts. The highest recovery of polyphenols (~8 mg GAE/g dry coffee solids) and reduction of hydroxymethylfurfural (95%) were obtained at 90 °C and 16% of ethanol during extraction and 80% of ethanol during purification. These operating conditions retained the antioxidant capacity of the crude extract between 60% and 88% depending on the determination method and recovered 90, 98, and 100% of 4-feruloylquinic acid, epicatechin, and 5-feruloylquinic acid, respectively after purification. The combined process allows differential polyphenols' recovery and enhances the safety of the extracts. Our computational chemistry results ruled out that the overall selectivity of the integrated process was correlated with the size of the polyphenols.

Determination of the Dissolution/Permeation and Apparent Solubility for Microencapsulated Emamectin Benzoate Using In Vitro and Ex Vivo Salmo salar Intestine Membranes.

In this work, two microencapsulation techniques were used to protect and improve the absorption of emamectin benzoate (EB), which is an antiparasitic drug used to control Caligus rogercresseyi. EB has a low aqueous solubility, which affects its absorption in the intestine of Salmo salar. Microparticles were produced by spray drying and ionic gelation, using Soluplus® (EB−SOL) and sodium alginate (EB−ALG) as polymers, respectively. Studies were conducted on dissolution/permeation, apparent permeability (Papp), apparent solubility (Sapp), and absorption using synthetic and biological membranes. Based on these results, the amount of EB in the microparticles needed to achieve a therapeutic dose was estimated. The EB−ALG microparticles outperformed both EB−SOL and free EB, for all parameters analyzed. The results show values of 0.45 mg/mL (80.2%) for dissolution/permeation, a Papp of 6.2 mg/mL in RS−L, an absorption of 7.3% in RS, and a Sapp of 53.1% in EM medium. The EB−ALG microparticles decrease the therapeutic dose necessary to control the parasite, with values of 3.0−2 mg/mL and 1.1−2 mg/mL for EB in EM and RS, respectively. The Korsmeyer−Peppas kinetic model was the best model to fit the EB−ALG and EB−SOL dissolution/permeation experiments. In addition, some of our experimental results using synthetic membranes are similar to those obtained with biological membranes, which suggests that, for some parameters, it is possible to replace biological membranes with synthetic membranes. The encapsulation of EB by ionic gelation shows it is a promising formulation to increase the absorption of the poorly soluble drug. In contrast, the spray-dried microparticles produced using Soluplus® result in even less dissolution/permeation than free EB, so the technique cannot be used to improve the solubility of EB.

Pressurized Hot Liquid Extraction with 15% v/v Glycerol-Water as An Effective Environment-Friendly Process to Obtain Durvillaea incurvata and Lessonia spicata Phlorotannin Extracts with Antioxidant and Antihyperglycemic Potential.

Brown seaweed phlorotannins have shown the potential to promote several health benefits. Durvillaea incurvata and Lessonia spicata-species that are widely distributed in central and southern Chile-were investigated to obtain phlorotannin extracts with antioxidant and antihyperglycemic potential. The use of an environmentally friendly and food-grade glycerol-based pressurized hot liquid extraction (PHLE) process (15% v/v glycerol water) was assessed for the first time to obtain phlorotannins. Multiple effects were analyzed, including the effect of the species, harvesting area (Las Cruces and Niebla), and anatomical part (holdfast, stipe, and frond) on the extracts' polyphenol content (TPC), antioxidant capacity (AC), and carbohydrate-hydrolyzing enzyme-α-glucosidase and α-amylase-inhibitory activity. Contaminants, such as mannitol, heavy metals (As, Cd, Pb, Hg, and Sn), and 5-hydroxymethylfurfural (HMF), were also determined. The anatomical part used demonstrated a significant impact on the extracts' TPC and AC, with holdfasts showing the highest values (TPC: 95 ± 24 mg phloroglucinol equivalents/g dry extract; DPPH: 400 ± 140 µmol Trolox equivalents/g dry extract; ORAC: 560 ± 130 µmol TE/g dry extract). Accordingly, holdfast extracts presented the most potent α-glucosidase inhibition, with D. incurvata from Niebla showing an activity equivalent to fifteen times that of acarbose. Only one frond and stipe extract showed significant α-glucosidase inhibitory capacity. No α-amylase inhibition was found in any extract. Although no HMF was detected, potentially hazardous cadmium levels (over the French limit) and substantial mannitol concentrations-reaching up to 50% of the extract dry weight-were found in most seaweed samples and extracts. Therefore, further purification steps are suggested if food or pharmaceutical applications are intended for the seaweed PHLE extracts obtained in this study.

Chemical Properties of Vitis Vinifera Carménère Pomace Extracts Obtained by Hot Pressurized Liquid Extraction, and Their Inhibitory Effect on Type 2 Diabetes Mellitus Related Enzymes.

Grape pomace polyphenols inhibit Type 2 Diabetes Mellitus (T2DM)-related enzymes, reinforcing their sustainable recovery to be used as an alternative to the synthetic drug acarbose. Protic co-solvents (ethanol 15% and glycerol 15%) were evaluated in the hot pressurized liquid extraction (HPLE) of Carménère pomace at 90, 120, and 150 °C in order to obtain extracts rich in monomers and oligomers of procyanidins with high antioxidant capacities and inhibitory effects on α-amylase and α-glucosidase. The higher the HPLE temperature (from 90 °C to 150 °C) the higher the total polyphenol content (~79%, ~83%, and ~143% for water-ethanol, water-glycerol and pure water, respectively) and antioxidant capacity of the extracts (Oxygen Radical Absorbance Capacity, ORAC), increased by ~26%, 27% and 13%, while the half maximal inhibitory concentration (IC50) decreased by ~65%, 67%, and 59% for water-ethanol, water-glycerol, and pure water extracts, respectively). Water-glycerol HPLE at 150 and 120 °C recovered the highest amounts of monomers (99, 421, and 112 µg/g dw of phenolic acids, flavanols, and flavonols, respectively) and dimers of procyanidins (65 and 87 µg/g dw of B1 and B2, respectively). At 90 °C, the water-ethanol mixture extracted the highest amounts of procyanidin trimers (13 and 49 µg/g dw of C1 and B2, respectively) and procyanidin tetramers of B2 di-O-gallate (13 µg/g dw). Among the Carménère pomace extracts analyzed in this study, 1000 µg/mL of the water-ethanol extract obtained, at 90 °C, reduced differentially the α-amylase (56%) and α-glucosidase (98%) activities. At the same concentration, acarbose inhibited 56% of α-amylase and 73% of α-glucosidase activities; thus, our grape HPLE extracts can be considered a good inhibitor compared to the synthetic drug.

Multi-response optimal hot pressurized liquid recovery of extractable polyphenols from leaves of maqui (Aristotelia chilensis [Mol.] Stuntz).

Multi-response optimization of hot pressurized liquid extraction (HPLE) was applied for the first time to obtain maqui (Aristotelia chilensis [Mol.] Stuntz) leaf extracts. The total polyphenol content (TPC), the antioxidant capacity (AC) as well as the total polyphenol purity of the maqui leaf extracts were accurately predicted (RSD < 8%) at the evaluated extraction scales. The optimum HPLE conditions that prioritized TPC and AC equally (OPT1) recovered ~3 times more TPC (205.14 mg GAE/g leaves) than maqui leaf extracts obtained by maceration, while the extract that prioritized purity over TPC and AC presented the highest purity (36.29%) and an EC50 ~3 times lower than currently reported values. It was found by multi-response optimization that maqui leaves and HPLE are among the best natural sources and extraction techniques, respectively, to recover protocatechuic acid, quercetin, and catechin.

Phlorotannins: From isolation and structural characterization, to the evaluation of their antidiabetic and anticancer potential.

Phlorotannins are phenolic characteristic compounds of brown seaweeds that are only constituted by phloroglucinol (1,3,5-trihydroxybenzene). They are chain- and net-like structures of diverse molecular weights and have been widely identified in Ecklonia, Eisenia, and Ishige species. Since the time they were discovered in the '70 s, phlorotannins have been suggested as a main factor responsible for the antimicrobial activities attributed to algae extracts. Currently, cumulative in vitro and in vivo research evidence the diverse bioactivities of phlorotannin extracts -such as antidiabetic, anticancer, and antibacterial- pointing out their potential pharmacological and food applications. However, metabolomic studies and clinical trials are scarce, and thus many phlorotannins health-beneficial effects in humans are not yet confirmed. This article reviews recent studies assessing the antidiabetic and anticancer activities of phlorotannins. Particularly, their potential to prevent and control the progression of these non-communicable diseases is discussed, considering in vitro and animal studies, as well as clinical interventions. In contrast to other approaches, we only included investigations with isolated phlorotannins or phlorotannin-rich extracts. Thus, phlorotannin extraction, purification and characterization procedures are briefly addressed. Overall, although considerable research showing the antidiabetic and anticancer potential of phlorotannins is now available, further clinical trials are still necessary to conclusively demonstrate the efficacy of these compounds as adjuvants for diabetes and cancer prevention or treatment.

Glycerol as Alternative Co-Solvent for Water Extraction of Polyphenols from Carménère Pomace: Hot Pressurized Liquid Extraction and Computational Chemistry Calculations.

Glycerol is a co-solvent for water extraction that has been shown to be highly effective for obtaining polyphenol extracts under atmospheric conditions. However, its efficacy under subcritical conditions has not yet been studied. We assessed different water-glycerol mixtures (15%, 32.5%, and 50%) in a hot pressurized liquid extraction system (HPLE: 10 MPa) at 90 °C, 120 °C, and 150 °C to obtain extracts of low molecular weight polyphenols from Carménère grape pomace. Under the same extraction conditions, glycerol as a co-solvent achieved significantly higher yields in polyphenols than ethanol. Optimal extraction conditions were 150 °C, with 32.5% glycerol for flavonols and 50% for flavanols, stilbenes, and phenolic acids. Considering gallic acid as a model molecule, computational chemistry calculations were applied to explain some unusual extraction outcomes. Furthermore, glycerol, methanol, ethanol, and ethylene glycol were studied to establish an incipient structure-property relationship. The high extraction yields of gallic acid obtained with water and glycerol solvent mixtures can be explained not only by the additional hydrogen bonds between glycerol and gallic acid as compared with the other alcohols, but also because the third hydroxyl group allows the formation of a three-centered hydrogen bond, which intensifies the strongest glycerol and gallic acid hydrogen bond. The above occurs both in neutral and deprotonated gallic acid. Consequently, glycerol confers to the extraction solvent a higher solvation energy of polyphenols than ethanol.

Functional fermented cherimoya (Annona cherimola Mill.) juice using autochthonous lactic acid bacteria.

The biochemical and functional properties of fermented Annona cherimola Mill. (cherimoya) juice using five lactic acid bacteria (LAB) isolated from autochthonous fruits from Northwestern Argentina were studied in this work. Fermentation was carried out at 30 °C for 48 h followed by a 21 day-storage period at 4 °C. The assayed LAB grew well during fermentation (final count of 108 CFU/mL, ΔpH ca. 1 U) and survived after the storage period. All strains consumed fructose and glucose present in cherimoya juice as energy sources, with the consequent synthesis of lactic and/or acetic acids as final metabolic products. However, only two of the five evaluated strains were capable to produce fermented cherimoya juices with a perceptible color change. Due to lactic acid fermentation, a moderate reduction in the total phenolic content (between 13% and 43%) was observed in the majority of the samples, although no change in the antioxidant capacity was detected. The fermented cherimoya juices showed a weak antiplatelet activity when adenosine diphosphate agonist was used. The findings of this study evidenced the potential use of Annona cherimola Mill. fermented juice as a novel matrix for the formulation of stable functional beverages with appealing nutritional and functional properties.

Polyphenols of Carménère Grapes.

Carménère is the emblematic grape of Chile. Recent studies indicate that it has a different polyphenolic profile than other commercial varieties of grape among other factors, due to its long maturation period. The grape and wine of Carménère stand out for having high concentrations of anthocyanins (malvidin), flavonols (quercetin and myricetin) and flavanols (catechin, epicatechin and epigallocatechin). These compounds are related to the distinctive characteristic of Carménère wine regarding astringency and color. In vivo and in vitro models suggest some positive effects of these polyphenols in the treatment and prevention of chronic diseases, such as atherosclerosis and cancer. Therefore, there is a high level of interest to develop scalable industrial methods in order to obtain and purify Carménère grape polyphenol extracts that could be used to improve the characteristics of wines from other varieties or produce nutraceuticals or functional foods for preventing and treating various chronic diseases.

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris.

BACKGROUND: Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions. RESULTS: In this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions. Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein. CONCLUSION: In this work, we formulated a dynamic genome scale metabolic model of Pichia pastoris that yields realistic metabolic flux distributions throughout dynamic cultivations. The model can be calibrated with experimental data to rationally propose genetic and process engineering strategies to improve the performance of a P. pastoris strain of interest.

Macromolecular Antioxidants and Dietary Fiber in Edible Seaweeds.

Seaweeds are rich in different bioactive compounds with potential uses in drugs, cosmetics and the food industry. The objective of this study was to analyze macromolecular antioxidants or nonextractable polyphenols, in several edible seaweed species collected in Chile (Gracilaria chilensis, Callophyllis concepcionensis, Macrocystis pyrifera, Scytosyphon lomentaria, Ulva sp. and Enteromorpha compressa), including their 1st HPLC characterization. Macromolecular antioxidants are commonly ignored in studies of bioactive compounds. They are associated with insoluble dietary fiber and exhibit significant biological activity, with specific features that are different from those of both dietary fiber and extractable polyphenols. We also evaluated extractable polyphenols and dietary fiber, given their relationship with macromolecular antioxidants. Our results show that macromolecular antioxidants are a major polyphenol fraction (averaging 42% to total polyphenol content), with hydroxycinnamic acids, hydroxybenzoic acids and flavonols being the main constituents. This fraction also showed remarkable antioxidant capacity, as determined by 2 complementary assays. The dietary fiber content was over 50% of dry weight, with some samples exhibiting the target proportionality between soluble and insoluble dietary fiber for adequate nutrition. Overall, our data show that seaweed could be an important source of commonly ignored macromolecular antioxidants.

Floral aroma improvement of Muscat spirits by packed column distillation with variable internal reflux.

The organoleptic quality of wine distillates depends on raw materials and the distillation process. Previous work has shown that rectification columns in batch distillation with fixed reflux rate are useful to obtain distillates or distillate fractions with enhanced organoleptic characteristics. This study explores variable reflux rate operating strategies to increase the levels of terpenic compounds in specific distillate fractions to emphasize its floral aroma. Based on chemical and sensory analyses, two distillate heart sub-fractions obtained with the best operating strategy found, were compared with a distillate obtained in a traditional alembic. Results have shown that a drastic reduction of the reflux rate at an early stage of the heart cut produced a distillate heart sub-fraction with a higher concentration of terpenic compounds and lower levels of negative aroma compounds. Therefore, this sub-fraction presented a much more noticeable floral aroma than the distillate obtained with a traditional alembic.

Aromatically enhanced pear distillates from blanquilla and conference varieties using a packed column.

Pear distillates are generally produced from the Bartlett variety because of its rich aroma. In this study, a chemical and sensorial comparative examination of pear distillates from the three main varieties grown in Spain (Bartlett, Blanquilla, and Conference) using two distillation systems (copper Charentais alembic and packed column) was undertaken. Volatile compounds were identified by gas chromatography to differentiate the spirits according to pear variety and distillation method. The Bartlett distillates from both distillation systems possessed higher ethyl ester and acetate and lower cis-3-hexen-1-ol and 1-hexanol concentrations. Despite these differences, a sensory analysis panel could distinguish only the Bartlett alembic distillate from the alembic distillates of the other varieties. In contrast, the panel rated the packed-column distillates equally. Therefore, less aromatic pear varieties can be used to produce distillates with aromatic characteristics similar to those of the Bartlett variety if a suitable distillation process is used.

Aromatic characterization of pot distilled kiwi spirits.

This study contributes fundamental knowledge that will help to develop a distillate of kiwi wine, made from kiwis of the Hayward variety grown in the southwest of Galicia (Spain). Two yeast strains, L1 (Saccharomyces cerevisiae ALB-6 from the EVEGA yeast collection) and L2 (S. cerevisiae Uvaferm BDX from Lallemand) were assessed to obtain a highly aromatic distillate. The kiwi spirits obtained were compared with other fruit spirits, in terms of higher alcohols, minor alcohols, monoterpenols, and other minor compounds, which are relevant in determining the quality and taste of the kiwi spirits. It was found that the kiwi juice fermented with yeast L1 produced a more aromatic distillate. In addition, kiwi distillates produced with both yeasts had the same ratio of trans-3-hexen-1-ol and cis-3-hexen-1-ol, which is lower than that found in other fruit distillates.