Pressurized Liquid (PLE) Truffle Extracts Have Inhibitory Activity on Key Enzymes Related to Type 2 Diabetes (α-Glucosidase and α-Amylase).

An optimized PLE method was applied to several truffle species using three different solvent mixtures to obtain bioactive enriched fractions. The pressurized water extracts contained mainly (1 → 3),(1 → 6)-ß-D-glucans, chitins, and heteropolymers with galactose and mannose in their structures. The ethanol extracts included fatty acids and fungal sterols and others such as brassicasterol and stigmasterol, depending on the species. They also showed a different fatty acid lipid profile depending on the solvent utilized and species considered. Ethanol:water extracts showed interesting lipids and many phenolic compounds; however, no synergic extraction of compounds was noticed. Some of the truffle extracts were able to inhibit enzymes related to type 2 diabetes; pressurized water extracts mainly inhibited the α-amylase enzyme, while ethanolic extracts were more able to inhibit α-glucosidase. Tuber brumale var. moschatum and T. aestivum var. uncinatum extracts showed an IC50 of 29.22 mg/mL towards α-amylase and 7.93 mg/mL towards α-glucosidase. Thus, use of the PLE method allows o bioactive enriched fractions to be obtained from truffles with antidiabetic properties.

Extraction and trapping of truffle flavoring compounds into food matrices using supercritical CO2.

A supercritical fluid extraction methodology was used to extract flavoring and bioactive compounds from truffles. Some parameters such as CO2 flow rate (1-3 mg/mL), extraction time (15-90 min) and different trapping food matrices (grape seed oil, gelatin, agar agar and water) were optimized using response surface methodology to enhance extraction and trapping yields. The optimal conditions (2.27 mg/mL CO2 flow rate, 82.5 min when using 40 °C and 30 MPa, with 1 mL grape seed oil as trapping matrix) obtained with Tuber melanosporum were applied to three different truffle species: Terfezia claveryi, Tuber aestivum and Tuber indicum. A total of 32 metabolites were profiled in the extracts using ultra-high-performance supercritical fluid chromatography coupled to quadrupole time-of-flight mass spectrometry. Compounds such as brassicasterol ergosta-7,22-dienol, oleic and linoleic acid were found at similar amounts in all the extracts but other molecules (e.g. fungal sterols) showed a particular distribution depending on the specie studied and whether a trapping matrix was used at the SFE outlet.

RNA-Seq, Bioinformatic Identification of Potential MicroRNA-like Small RNAs in the Edible Mushroom Agaricus bisporus and Experimental Approach for Their Validation.

Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.

Application of Pressurized Liquid Extractions to Obtain Bioactive Compounds from Tuber aestivum and Terfezia claveryi.

A PLE (pressurized liquid extraction) method was adjusted following a full-factorial experimental design to obtain bioactive-enriched fractions from Tuber aestivum and Terfezia claveryi. Temperature, time and solvent (water, ethanol and ethanol-water 1:1) parameters were investigated. The response variables investigated were: obtained yield and the levels of total carbohydrate (compounds, ß-glucans, chitin, proteins, phenolic compounds and sterols). Principal component analysis indicated water solvent and high temperatures as more adequate parameters to extract polysaccharide-rich fractions (up to 68% of content), whereas ethanol was more suitable to extract fungal sterols (up to 12.5% of content). The fractions obtained at optimal conditions (16.7 MPa, 180 °C, 30 min) were able to protect Caco2 cells from free radical exposure, acting as antioxidants, and were able to reduce secretion of pro-inflammatory cytokines in vitro: IL-6 (50%), and TNFα (80% only T. claveryi ethanol extract), as well as reduce high inhibitory activity (T. aestivum IC50: 9.44 mG/mL).

Modulation of human intestinal microbiota in a clinical trial by consumption of a ß-D-glucan-enriched extract obtained from Lentinula edodes.

PURPOSE: The aim of this study was to evaluate the hypocholesterolemic, immune- and microbiota-modulatory effect of a mushroom extract in hypercholesterolemic subjects. METHODS: A randomized, controlled, double-blind, and parallel clinical trial was carried out with subjects from 18 to 65 years old (n = 52) with untreated mild hypercholesterolemia. Volunteers consumed a ß-D-glucan-enriched (BGE) mixture (10.4 g/day) obtained from shiitake mushrooms (Lentinula edodes) ensuring a 3.5 g/day of fungal ß-D-glucans or a placebo incorporated in three different commercial creams. RESULTS: This mixture showed hypocholesterolemic activities in vitro and in animal studies. After eight weeks intervention, no significant differences in lipid- or cholesterol-related parameters were found compared to placebo subjects as well as before and after the BGE mixture administration. No inflammatory or immunomodulatory responses were noticed and no changes in IL-1ß, IL-6, TNF-α or oxLDL were recorded. However, consumption of the BGE mixture was safe and managed to achieve the dietary fibre intake recommended as cardiovascular protective diet. Moreover, the BGE mixture modulated the colonic microbiota differently compared to placebo. Microbial community composition varied from before to after the intervention with several genera being positively or negatively correlated with some biomarkers related to cholesterol metabolism. CONCLUSION: These results suggested a relation between cholesterol metabolism, microbiota and BGE administration. Nevertheless, the precise significance of this differential modulation was not fully elucidated and requires further studies.

Effects of gamma irradiation on the shelf-life and bioactive compounds of Tuber aestivum truffles packaged in passive modified atmosphere.

The effects of gamma irradiation (0.5, 1.0, 1.5 and 2.5 kGy doses) on Tuber aestivum packaged under modified atmosphere was evaluated. The respiration rate, microbial populations, sensory characteristics and content of bioactive compounds (total carbohydrates, chitins, ß-glucans, proteins, total phenols and sterols) were monitored from immediately after treatment up to day 42 of storage at 4 °C. All the irradiation treatments tested reduced the microbial groups studied by more than 3 log cfu/g. Increasing irradiation doses slowed down the subsequent microbial development throughout the conservation period for all the groups studied. The irradiation treatments did not negatively affect truffle sensory characteristics. Only a slight visible superficial yeast growth was detected at the end of the shelf-life in all doses applied. Total carbohydrate content, chitins, ß-glucans and proteins levels were not affected after irradiation. However, sterols, particularly stigmasterol, slightly decreased after irradiation, while levels of phenolic compounds doubled during storage. Gamma irradiation (2.5 kGy) could be used to extend the shelf-life of summer truffles packaged under modified atmosphere, since no remarkable reduction of bioactive compounds were noticed after 42 days of storage, and their sensory and microbial parameters were of higher quality than those of non-irradiated controls.

Screening of bioactive compounds in truffles and evaluation of pressurized liquid extractions (PLE) to obtain fractions with biological activities.

Truffles, besides the appreciated aromatic compounds, contain other molecules with interesting bioactive properties. A screening of fungal sterols and ß-glucans within different truffle species and locations was carried out. These compounds were extracted with pressurized liquids (PLE) generating enriched fractions. Extraction efficiency was studied with a full-factorial experimental design (Response surface methodology, RSM), using water and ethanol as extraction solvents. Polysaccharides from truffle powder (TP) and the optimal PLE extract (EP) obtained were precipitated and analysed by NMR and GC-MS. THP-1 cell cultures were utilized to test immunomodulatory properties. With the optimal PLE conditions (16.7 MPa, 180 °C, 30 min) 64 and 22.5% yields were obtained respect, with water and ethanol, generating fractions containing respect, 9.1% ß-glucan and 4.5% ergosterol. NMR analyses detected (1 â†’ 3)-ß-glucan structures in truffle. The EP induced a reduction of 40% IL-1ß and 60% IL-6 pro-inflammatory cytokines secretion suggesting potential immunomodulatory activity.

Effect of tartrazine on digestive enzymatic activities: in vivo and in vitro studies.

Tartrazine (E102) is a synthetic food coloring, which belongs to the class of mono azo dyes and is known to cause numerous health problems. The current research aimed to evaluate the effect of this food dye on the enzymatic activity of amylase, lipase and proteases after a subchronic ingestion in Swiss mice. Additionally, an in vitro digestion model was used to highlight the relationship between the probable toxicity of tartrazine and the nature of the food ingested. The results show that there were no adverse effects of tartrazine on the body weight gain, and on amylase or lipase activities. However, in the high dose of tartrazine (0.05%) group, a significant decrease in trypsin and chymotrypsin enzymatic activities were observed. Regarding the in vitro digestion model, our findings show that there were no changes in the trypsin and chymotrypsin enzymatic activities either using 7.5 or 75 mg of tartrazine mixed with rice, butter or milk. We conclude that excessive consumption of tartrazine appears to alter the enzymatic activity of proteases in vivo which may have deleterious consequences on digestion. Even thought the dose close to the acceptable daily intake does not affect those activities, a strict control of tartrazine dose in high-consumption foods especially among children is an indispensable task.

Isolation and comparison of α- and ß-D-glucans from shiitake mushrooms (Lentinula edodes) with different biological activities.

A polysaccharide-enriched extract obtained from Lentinula edodes was submitted to several purification steps to separate three different D-glucans with ß-(1→6), ß-(1→3),(1→6) and α-(1→3) linkages, being characterized through GC-MS, FT-IR, NMR, SEC and colorimetric/fluorimetric determinations. Moreover, in vitro hypocholesterolemic, antitumoral, anti-inflammatory and antioxidant activities were also tested. Isolated glucans exerted HMGCR inhibitory activity, but only ß-(1→6) and ß-(1→3),(1→6) fractions showed DPPH scavenging capacity. Glucans were also able to lower IL-1ß and IL-6 secretion by LPS-activated THP-1/M cells and showed cytotoxic effect on a breast cancer cell line that was not observed on normal breast cells. These in vitro results pointed important directions for further in vivo studies, showing different effects of each chemical structure of the isolated glucans from shiitake mushrooms.

In vitro and in vivo testing of the hypocholesterolemic activity of ergosterol- and ß-glucan-enriched extracts obtained from shiitake mushrooms (Lentinula edodes).

Herein, a supercritical extraction plant (with a 6 L extraction cell) was successfully used to obtain ergosterol-enriched extracts from Lentinula edodes under the following conditions: a temperature of 40 °C, pressure of 225 bar, reaction time of 1-5 h, and the flow rate of 20 L h-1 for recirculated CO2. Moreover, ergosterol (ERG) and the SFE extract (SFE) with highest ergosterol concentration were microemulsified and submitted to in vitro digestion to study their ability to displace cholesterol from dietary mixed micelles (DMMs). ERG was also mixed with a ß-glucan-enriched (33.5%) extract (BGE) obtained from L. edodes to investigate the synergies between them; the results indicated that all these extracts (including BGE without ERG) could reduce the cholesterol levels in the DMMs. However, when ERG and SFE were simultaneously administered to mice with a hypercholesterolemic diet, no significant differences in the serum cholesterol levels were detected as compared to the case of the control. However, when only BGE was administrated to another mice model previously induced with hypercholesterolemia, significant reduction in the cholesterol levels was noticed.

Strengths and weaknesses of the aniline-blue method used to test mushroom (1→3)-ß-d-glucans obtained by microwave-assisted extractions.

The parameters to extract polysaccharide-enriched fractions (PEF) from mushrooms using MAE (microwave-assisted extraction) were adjusted following a full factorial 32 experimental design. The highest yield and total carbohydrate values, using Lentinula edodes as model mushroom, were obtained at 180 °C and 30 min. Several mushroom species were submitted to MAE and their PEF yields ranged between 12.1-44.2%. (1→3)-ß-Glucans determination using a conventional fluorimetric method changed depending on the standard utilized. NMR analyses of PEF indicated that the presence of other polysaccharides in the extracts or their specific folding, might impair the proper determination of (1→3) linkages by the fluorophore. Mushrooms from Cantharellales order contained (1→3)-ß-glucans but they were not detected with the fluorimetric method. Therefore, although the method (after adjustments) was sensitive enough to detect their presence in many mushroom extracts, it cannot be used for all species and it is also not recommended for quantitative determinations.

Effect of traditional and modern culinary processing, bioaccessibility, biosafety and bioavailability of eritadenine, a hypocholesterolemic compound from edible mushrooms.

Eritadenine is a hypocholesterolemic compound that is found in several mushroom species such as Lentinula edodes, Marasmius oreades, and Amanita caesarea (1.4, 0.7 and 0.6 mg per g dry weight, respectively). It was synthesized during all developmental stages, being present in higher concentrations in the skin of shiitake fruiting bodies. When subjected to traditional cooking, grilling followed by frying were more adequate methodologies than boiling or microwaving to maintain its levels. Modern culinary processes such as texturization (with agar-agar) and spherification (with alginate) also interfered with its release. Grilling and gelling using gelatin enhanced eritadenine's bioaccessibility in an in vitro digestion model. An animal model (where male and female rats were administered 21 and 10 mg per kg animal per day of eritadenine) indicated that intake of the compound was safe under these concentrations; it reached the liver and reduced the atherogenic index (TC/HDL) in rat sera. Thus, it might be used to design a functional food.

Document of recommendations of the SEA 2018. Lifestyle in cardiovascular prevention. / Documento de recomendaciones de la SEA 2018. El estilo de vida en la prevención cardiovascular.

Lifestyle is a complex concept that includes aspects external to ourselves that can modulate and influence our health. The knowledge of the relationship between lifestyle and cardiovascular risk does not attain the level of evidence achieved with clinical trials with drugs, because clinical studies are scarce and mainly of observational nature, albeit based on large cohorts. Nutritional epidemiology has the added difficulty of being based mostly on subjective dietary recall methods to ascertain nutrient and food intake over time, with the additional problems of incomplete data collection, variable measurements of adherence due to seasonal and geographical differences in food composition, and the changing eating behavior that human beings have over time. The purpose of this document is to carry out an updated and hierarchical review of the relationship between lifestyle and cardiovascular disease based on current evidence, paying attention to three aspects that are of great pathogenic importance and are directly modifiable: physical activity, tobacco consumption, and diet. With this, we intend to update the knowledge on this relationship, construct evidence-based recommendations, and provide a simple tool for clinical practice especially directed to health professionals involved in the care of people at cardiovascular risk, defining simple and easy strategies for individuals who receive advice for the primary and secondary prevention of cardiovascular diseases.

Extraction of bioactive compounds against cardiovascular diseases from Lentinula edodes using a sequential extraction method.

Three extraction methods were sequentially combined to obtain fractions from Lentinula edodes (shiitake mushrooms) containing bioactive compounds against cardiovascular diseases (CVDs). Fruiting bodies were first extracted with plain water, obtained residue was then submitted to supercritical fluid extraction (SFE) and remaining residue submitted to hot water extraction. Sequential design allowed reutilization of the nonextracted material as raw material for the successive extractions increasing extraction yields and separating interesting compounds. Obtained fractions contained different amounts of ß-glucans, chitins, eritadenine, lenthionine, ergosterol, proteins/peptides and phenolic compounds conferring them different bioactivities. Water soluble fractions showed high antioxidant activities (ABTS+• and DPPH• scavenging capacity and reducing power), they were also able to inhibit one of the main enzymes involved in hypertension (angiotensin-I converting enzyme) and the key enzyme of cholesterol metabolism (3-hydroxy-3-methylglutaryl coenzyme A reductase). The latter inhibitory activity was also noticed in SFE extracts although ergosterol and other lipid-like molecules were isolated. Dietary fibers were separated in the third extraction. Therefore, with this sequential extraction procedure bioactive compounds against CVDs can be selectively separated from a single batch of shiitake powder. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:746-755, 2018.

Molecular actions of hypocholesterolaemic compounds from edible mushrooms.

Cholesterol levels are strictly regulated to maintain its homeostasis; therefore, if it is not absorbed with the diet, the cholesterol biosynthetic pathway is enhanced and vice versa. Nowadays, the commonly prescribed therapeutic treatments for hypocholesterolemic patients are targeted toward the reduction of both cholesterol intestinal absorption and/or its endogenous biosynthesis. But, when hypercholesterolemia is still moderate the consumption of food products with cholesterol-lowering capacities is more desirable than using drugs. Marketed foods supplemented with hypocholesterolemic compounds are only inhibiting mechanisms for cholesterol absorption (i.e. phytosterols and cereal ß-glucans). However, certain fungal extracts obtained from edible mushrooms might be able to modulate cholesterol levels by both strategies, pharmaceutical drugs and functional foods. In vitro and in vivo studies indicated that fungal sterols down-regulated genes involved in cholesterol homeostasis (such as Srebf2 and Nr1h4 (FXR)) and other specific mushroom extracts (ß-glucans and other water-soluble compounds) also stimulated transcriptional profiles similar to simvastatin or ezetimibe (two hypocholesterolemic drugs). These and other observations suggested that the hypocholesterolemic effect of mushroom extracts could be due to transcriptional and post-transcriptional modulations besides other indirect effects.

Water-Soluble Polysaccharide Extracts from the Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) with HMGCR Inhibitory Activity.

Water extracts from Pleurotus ostreatus containing no statins showed 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitory activity (in vitro) that might be due to specific water-soluble polysaccharides (WSPs); when isolated and deproteinized, increasing concentrations of the WSP extract induced higher inhibition. The WSP extract contained mainly ß-glucans, mannogalactans, and glycogen (e.g., α-glucans), although derivatives or fragments with lower molecular weights (between 14 and 3.5 kDa) were present and were able to induce the inhibitory activity. The extract contained more ß-(1→3)-glucans than ß-(11→3),(11→6)-glucans, and they partially survived digestion and managed to pass through Caco2 cell monolayers to the lower compartment after in vitro digestion and transport experiments. The WSP might also modulate Caco2 membrane integrity.

Evaluation of microwave-assisted and pressurized liquid extractions to obtain ß-d-glucans from mushrooms.

Microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE) were compared as advanced technologies to obtain polysaccharides (particularly biologically active ß-glucans) from Pleurotus ostreatus and Ganoderma lucidum fruiting bodies. Extraction effectiveness was compared by a full-factorial experimental design (response surface methodology, RSM), using water as extraction solvent. Total carbohydrate content of the obtained extracts and polysaccharide yields were the variable responses investigated, while temperature and extraction time were the experimental factors. Temperature showed stronger influence in the polysaccharide extraction than time. The latter factor slightly affected MAE but not PLE extractions. Optimal conditions within the studied range were determined for each extraction method and species based on the desirability functions. Regarding the polysaccharide composition, the main differences between the species were more quantitative rather than qualitative, since NMR analyses indicated that all extracts contained mainly ß- and α-glucans and heteropolysaccharides. Both extraction systems were effective for polysaccharide extraction from mushrooms.

Plasma Cholesterol-Lowering Activity of Lard Functionalized with Mushroom Extracts Is Independent of Niemann-Pick C1-like 1 Protein and ABC Sterol Transporter Gene Expression in Hypercholesterolemic Mice.

Interest in food matrices supplemented with mushrooms as hypocholesterolemic functional foods is increasing. This study was to (i) investigate the hypocholesterolemic activity of lard functionalized with mushroom extracts (LF) including fungal ß-glucans, water-soluble polysaccharides, or ergosterol and (ii) examine the LF influence on transcriptional mechanisms involved in cholesterol metabolism. mRNA levels of 17 cholesterol-related genes were evaluated in jejunum, cecum, and liver of high cholesterol-fed mice. The four tested LFs decreased plasma cholesterol by 22-42%, HDLc by 18-40%, and LDLc by 27-51%, and two of them increased mRNA levels of jejunal Npc1l1 and Abcg5 and hepatic Npc1l1. mRNA levels of other cholesterol-related genes were unchanged. These findings suggest that LF may have potential as a dietary supplement for counteracting diet-induced hypercholesterolemia and could be a source for the development of novel cholesterol-lowering functional foods. However, the cholesterol-lowering effect was unrelated to transcriptional changes, suggesting that post-transcriptional mechanisms could be involved.

Water-Soluble Compounds from Lentinula edodes Influencing the HMG-CoA Reductase Activity and the Expression of Genes Involved in the Cholesterol Metabolism.

A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and ß-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.

Modulation of cholesterol-related gene expression by ergosterol and ergosterol-enriched extracts obtained from Agaricus bisporus.

PURPOSE: To investigate the effect of two extracts obtained from Agaricus bisporus on the mRNA expression of cholesterol-related genes. One of the extracts contained ergosterol and other fungal sterols (SFE) and the other contained ß-glucans and fungal sterols (EßG). METHODS: Firstly, the dietary mixed micelles (DMMs) generated after in vitro digestion of standards and SFE were applied to Caco2 cells. Then, the lower compartment after a Caco2-transport experiment was applied to HepG2 cells. The mRNA expression was assessed in both cell lines by low-density arrays (LDA). Mice received the extracts, ergosterol or control drugs after 4 weeks of a high-cholesterol diet. The lipid profile of plasma, liver and feces was determined. LDA assays were performed in liver and intestines. RESULTS: The DMM fraction of SFE up-regulated the LDLR mRNA expression in Caco2 cells. The lower compartment after Caco2-transport experiments up-regulated LDLR and modulated several other lipid-related genes in HepG2 cells. In mice, SFE decreased TC/HDL ratio and reduced hepatic triglycerides paralleled with down-regulation of Dgat1 expression, while EßG did it without transcriptional changes. Addition of SFE or ergosterol induced in jejunum a similar transcriptional response to simvastatin and ezetimibe; they all down-regulated Srebf2 and Nr1h4 (FXR) genes. CONCLUSION: Ergosterol-containing extracts from A. bisporus lowered hepatic triglyceride and modify the mRNA expression of cholesterol-related genes although the transcriptional regulation was unrelated to changes in plasma lipid profile. These extracts may be useful limiting hepatic steatosis and as bioactive ingredients to design novel functional foods preventing lifestyle-related diseases such as non-alcoholic fatty liver disease.