Isolation, characterization and HepG-2 inhibition of a novel proteoglycan from Flammulina velutipes.

Flammulina velutipes has anti-inflammatory, immunomodulatory, antioxidant and many bioactive properties with high contents of carbohydrate, proteins and fibers. In this study, a novel proteoglycan with polysaccharide complexes and protein chain, named PGD1-1, was isolated from F. velutipes. The structural characteristics of PGD1-1 were then determined, and its anti-proliferation and pro-apoptotic activities against HepG-2 cells were demonstrated in vitro. Results proved that the average molecular weight of PGD1-1 was 32.71 kDa, and the carbohydrate and protein contents were 93.35 and 2.33%, respectively. The protein moiety was bonded to a polysaccharide chain via O-glycosidic linkage. The monosaccharides consisted of d-glucose, D-galactose and D-xylose in a molar ratio of 21.90:2.84:1.00. PGD1-1 significantly inhibited the proliferation of HepG-2 cells by affecting cell lipid peroxidation and nitric oxide production. In addition, PGD1-1 promoted the apoptosis of HepG-2 cells, especially the early apoptosis. These findings proved that PGD1-1 was a novel potent ingredient against the proliferation of HepG-2, which will provide a theoretical basis for the development and utilization of the functional ingredients of the F. velutipes.

Isolation, purification and identification of immunologically active peptides from Hericium erinaceus.

Biologically active peptides released by proteins are important in regulating immunity. The purpose of this study was to isolate and purify an immunologically active peptide from Hericium erinaceus (H. erinaceus) and to explore its effect on cytokine secretion and differentiation of macrophages. An active peptide with an amino acid sequence, Lys-Ser-Pro-Leu-Tyr (KSPLY) was obtained from H. erinaceus protein by ultrafiltration combined with multistage chromatography separation and identification technology. Subsequently, it was confirmed that the synthetic peptide KSPLY had a good immunomodulatory activity at a concentration of 100 µmol/L and could promote the secretion of NO, IL-1ß, IL-6 and TNF-α by macrophages. The effects of KSPLY on M1 macrophages and M2 macrophages were also studied. Results showed that KSPLY inhibited the secretion of NO and IL-6 by M1 macrophages and promoted the tendency of M2 macrophages to transform to M1 macrophages. Therefore, it can be concluded that KSPLY is an effective immunomodulatory peptide that may be beneficial in cancer treatment and human health improvement.

Nanocomposite packaging regulates extracellular ATP and programed cell death in edible mushroom (Flammulina velutipes).

Our previous study indicated that nanocomposite packaging material (Nano-PM) containing nano-Ag, nano-TiO2, nano-SiO2 and nanoattapulgite alleviated postharvest senescence of Flammulina velutipes by regulating respiration and energy metabolism. In this study, extracellular ATP (eATP) and programmed cell death (PCD) were employed as critical factors to further investigate the senescence mechanism of postharvest F. velutipes. Results demonstrated that Nano-PM delayed apyrase activity decrease and stimulated critical oxidative phosphorylation-related gene expression to inhibit eATP content increase, which is a crucial signaling molecule related to delaying senescence. The regulation of eATP resulted in alleviating PCD including chromosomal concentration, DNA fragmentation, Ca2+ influx, high caspase-1 activity and cytochrome c content and leading to high cell viability. Overall, Nano-PM alleviated PCD and postharvest senescence of F. velutipes by regulating extracellular ATP.

Exogenous bacterial composition changes dominate flavor deterioration of dried carrots during storage.

Flavor deterioration is a serious problem in dried carrots during storage and is frequently accompanied by water absorption and bacterial growth. To explore the underlying mechanism of flavor deterioration, relationship among water status, exogenous bacterial composition and flavor changes in dried carrots were analyzed at different water activities (aw, 0.43, 0.67, 0.76 and 0.84). Results suggested that the water molecules mobility significantly increased in the dried carrots at higher aw levels (0.67, 0.76 and 0.84), this was attributed to the raised content of bound water, rather than immobilized or free water. Consequently, this accelerated microbial growth and flavor deterioration. At aw = 0.84, the characteristic flavor compounds including 2,3-butanediol, pentanoic acid, hexanoic acid, heptanoic acid and nonanoic acid were lost. The disagreeable flavor compounds including terpenes were produced during the storage period. These were the main contributors of flavor deterioration in the dried carrots. Lactic acid bacteria, as the dominant bacteria in dried carrots during storage, were proved to be closely related to the production of o-cymene, ß-pinene and ß-myrcene. Moreover, the emergence of Pediococcus spp. was the major factor leading to the increase of γ-terpinene in dried carrots.

Ultrahigh-Pressure Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry-Based Metabolomics Reveal the Mechanism of Methyl Jasmonate in Delaying the Deterioration of Agaricus bisporus.

Conquering rapid postripeness and deterioration of Agaricus bisporus is quite challenging. We previously observed that methyl jasmonate (MeJA) pretreatment postponed the deterioration of A. bisporus, but the mechanism is unknown. Here, a nontargeted metabolomics analysis by ultrahigh-pressure liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) revealed that MeJA increased the synthesis of malate by inhibiting the decomposition of fumarate and cis-aconitate. MeJA maintained energy supply by enhancing ATP content and energy charge level and improving hexokinase and glucose-6-phosphate dehydrogenase activities as well. These results promoted ATP supply by maintaining glycolysis, the TCA cycle, and the pentose phosphate pathway. In addition, we revealed that the delayed deterioration was attributed to MeJA treatment which stimulated the energy status of A. bisporus by reducing the respiration rate and nutrient decomposition, thus maintaining energy production. Our results provide a new insight into the role of MeJA treatment in delaying deterioration of A. bisporus through ATP production and supply.

Mass transfer characteristics during ultrasound-assisted osmotic dehydration of button mushroom (Agaricus bisporus).

Button mushroom (Agaricus bisporus) slices were dehydrated using ultrasound-assisted osmotic dehydration (UOD), and three osmotic agents including sucrose, glucose and sodium chloride were investigated for their effects on the mass transfer characteristics, average density and microstructure. Different mathematical models were selected to describe the osmotic behaviours, and the effective moisture (D m) as well as solid diffusivities (D s) during UOD were also calculated. The results showed that, during UOD, button mushrooms had the highest D m values in the sodium chloride solutions, and they had the highest D s values in the glucose solutions, which indicated that faster moisture and solid transfers could occur in these two osmotic solutions, respectively. Moreover, the Weibull model provided the best fit for the UOD curves of button mushrooms under the study's operating conditions, which showed good predictability for the moisture and solid contents of the button mushrooms during UOD. In addition, sucrose agents were suggested for use in the UOD of button mushrooms due to the better microstructure of the products as well as the appropriate rates of effective moisture and solid diffusivities during UOD. This study provides a theoretical basis for the deep processing of mushrooms and other food products.

Variation of volatile terpenes in the edible fungi mycelia Flammulina velutipes and communications in fungus-mite interactions.

Many mites rely on fungi for nutrients, and fungi benefit from them with regard to spore dispersal, or nutrient resources. The interactions among mites and fungi are still not clear in most cases. This study analyzed volatile natural products from the liquid and solid cultures of the edible fungi, Flammulina velutipes (Fr.) Sing, and the solid mycelia induced by the storage mite, Tyrophagus putrescentiae Schrank, using HS-SPME-GC-MS/MS. Five new monoterpenes and 30 new sesquiterpenes were isolated from the two cultures of F. velutipes and a newly monoterpene and 14 newly sesquiterpenes found in the solid mycelia induced by the storage mite. Sesquiterpenes were abundant in the mycelial stage of F. velutipe. The mite was attracted by some volatiles from host fungi, dihydrocarveol, cedrol, ß-caryophyllene, α-terpilene, ß-pinene and benzaldehyde, analyzed by four-arm olfactometer. Some terpenes induced by T. putrescentiae, such as caryophyllene oxide, bicyclogermacrene, and (-)-spathulenol, would have potential biological function. These results suggest that some volatile sesquiterpenes play an important role in enabling the mite to recognize host fungi.

Effect of nanocomposite packaging on postharvest senescence of Flammulina velutipes.

A polyethylene based packaging material containing nano-Ag, nano-TiO2, nano-SiO2, and attapulgite has been prepared. The effect of nanocomposite packaging material (Nano-PM) on the senescence of Flammulina velutipes during 15 days of postharvest storage at 4 °C and a relative humidity of 90% were analyzed. The results showed that compared with normal packaging material (Normal-PM) and no packaging (No-PM), Nano-PM improved the appearance quality, reduced weight loss and cap opening. The degree of maturity and increase in molecular weight of F. velutipes polysaccharides (FVP) were delayed. The content loss of proteoglycan protein was less and degree of oxidation was lower. The storage with Nano-PM reduced the fibrosis of texture, cellulase activity, the accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2-) by 18.9%, 48.3%, 26.6% and 27.8%, respectively (P < .05). The Nano-PM effectively delayed the postharvest senescence of F. velutipes, hence prolonged its shelf life and increased its preservation quality.

Effect of hot air drying on volatile compounds of Flammulina velutipes detected by HS-SPME-GC-MS and electronic nose.

Volatile compounds are important factors that affect the flavor quality of Flammulina velutipes, but the changes occurring during hot air drying is still unclear. To clarify the dynamic changes of flavor components during hot air drying, comprehensive flavor characterization and volatile compounds of F. velutipes were evaluated using electronic nose technology and headspace solid phase micro-extraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS), respectively. Results showed that volatile components in F. velutipes significantly changed during hot air drying according to the principal component analysis and radar fingerprint chart of electronic nose. Volatile compounds of fresh F. velutipes consisted mainly of ketones, aldehydes and alcohols, and 3-octanone was the dominant compound. Drying process could significantly decrease the relative content of ketones and promoted the generation of alcohols, acids, and esters, which became the main volatile compounds of dried F. velutipes. These may provide a theoretical basis for the formation mechanism of flavor substances in dried F. velutipes.

Separation and speciation analysis of zinc from Flammulina velutipes.

Orthogonal experiment was applied to optimize the water extraction parameters of zinc from Flammulina velutipes, and then the extracts were separated by membrane filter (0.45 µm) and D101 macroporous resin. Six different species of Zn were obtained and the Zn content of various species were determined by flame atomic absorption spectrometry. The optimized conditions for the extraction of Zn were: ratio of dried material to water, 1:30; extraction temperature, 75°C; extraction time, 120 minutes. About 34.43 µg Zn was extracted from 1 g dried F. velutipes powder under the optimal conditions. The recovery value for Zn was 96.5% with a low relative standard deviation. In addition, the content of the organic state of Zn was more than that of the inorganic state, and most of the organic state Zn was found in the polysaccharide and protein fractions.

Purification, characterization and antitumor activity of polysaccharides from Pleurotus eryngii residue.

A novel water-soluble polysaccharide from Pleurotus eryngii residue was isolated and further purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography to yield PEPE-1, PEPE-2 and PEPE-3. Molecular weights were determined by high-performance size-exclusion chromatography (HPSEC). Gas chromatography (GC) analysis of monosaccharide composition confirmed that PEPE-1, PEPE-2 and PEPE-3 were heteropolysaccharides and mainly composed of glucose. Sulfate and uronic acid content, ultraviolet and infrared spectrum were also evaluated. The antitumor activities of the polysaccharides against HepG-2 cells were studied in vitro. Results showed that the three polysaccharides could suppress the proliferation and enhance lactate dehydrogenase (LDH) release of HepG-2 cells in a dose- and time-dependent manner. The effect increased in the order of PEPE-1

Changes in non-volatile taste components of button mushroom (Agaricus bisporus) during different stages of freeze drying and freeze drying combined with microwave vacuum drying.

Button mushroom slices were dehydrated using freeze drying (FD) or FD combined with microwave vacuum drying (FMVD), and the non-volatile component profiles were studied. The results showed that the level of non-volatile components in button mushroom firstly increased during sublimation of FD/FMVD process and then fell during desorption in FD process and MVD in FMVD process. Compared to FD products, the contents of soluble sugars and polyols in FMVD products were relatively low, whereas the contents of total free amino acids were significantly higher, close to the level of fresh mushroom. However, there was no significant difference in the contents of 5'-nucleotides and organic acids between FD and FMVD products. The equivalent umami concentration (EUC) values for FD and FMVD products did not differ from fresh, indicating that both drying methods could effectively preserve MSG (monosodium glutamate)-like components in button mushroom.

Immunopotentiation of Pleurotus eryngii (DC. ex Fr.) Quel.

ETHNOPHARMACOLOGICAL RELEVANCE: Pleurotus eryngii (DC. ex Fr.) Quel has been collected from the wild, cultivated and used in traditional medicines to treat various disorders and diseases since antiquity. In traditional Chinese medicine, the powdered fruiting bodies of Pleurotus eryngii were used for immunostimulation, skin-care, wound-healing, cancer and lumbago treatment. In the current study, we investigated the antiproliferative activity of Pleurotus eryngii powder on A549, BGC-823, HepG2 and HGC-27 cancer cells and its immunomodulating activity on macrophage, RAW 264.7 cells based on its active compound. MATERIALS AND METHODS: A novel bioactive protein (PEP) was extracted from Pleurotus eryngii fruiting bodies powder and purified on DEAE-52, CM-52 and Superdex 75 column chromatographies using an ÄKTA purifier. Its cytotoxicity on A549, BGC-823, HepG2, HGC-27 and RAW 267.4 cell lines was then evaluated using MTT, alamar blue (AB), trypan blue (TB), neutral red (NR), lactate dehydrogenase (LDH), Annexin V FITC/PI and morphological change assays. Moreover, lysosomal enzyme activity, pinocytosis, nitric oxide (NO) and hydrogen peroxide (H2O2) production assays were used to examine immunomostimulatory activity of PEP on RAW 267.4 cells. RESULTS: Based on high performance gel permeation chromatography (HPGPC), Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) analyses, the isolated protein (PEP) had a molecular weight of 63 kDa, a secondary (α-helical) structure and was mainly composed of arginine, serine and glycine. PEP significantly (P<0.05) inhibited A549, BGC-823, HepG2 and HGC-27 tumor cells proliferation dose-dependently with an IC50 range of 36.5 ± 0.84 to 229.0 ± 1.24 µg/ml. Contrarily, PEP stimulated the proliferation of macrophages. CONCLUSION: Pleurotus eryngii fruiting bodies powder has a potential application as a natural antitumor agent with immunomodulatory activity, proposedly, by targeting the lysosomes of cancerous cells and stimulating macrophage-mediated immune responses.

Antiproliferative and immunostimulatory activity of a protein from Pleurotus eryngii.

BACKGROUND: Currently, the use of mushrooms as functional foods, nutraceuticals or phytopharmaceuticals source has risen. In contrast, the possible cellular cytotoxicity and immunostimulatory activity of Pleurotus eryngii (DC. ex Fr.) Quel protein (PEQP) is unknown. Here we report extraction, anti-tumorigenic and immunostimulatory activity of PEQP in vitro. RESULTS: PEQP was extracted from the dried fruiting bodies of P. eryngii, purified and characterised. Its in vitro antiproliferative activity was then evaluated in human non-small cell lung cancer A549 (NSCLC), stomach adenocarcinoma BGC-823, hepatocellular carcinoma HepG2 and gastric carcinoma HGC-27 cell lines using conventional cancer drugs (paclitaxel, doxorubicin and mitomycin C) as positive controls. The protein fractions (PEQP 1, 2, 3 and 4) obtained inhibited tumour cell proliferation dose-dependently with fraction PEQP 2 having significant (P < 0.05) toxicity in all tumour cells. PEQP had no significant toxicity on normal liver Chang cells but their proliferation was significantly inhibited by mitomycin C. Moreover, PEQP stimulated the proliferation, lysosomal enzyme activity, pinocytosis, nitric oxide and hydrogen peroxide production of RAW 264.7 cell lines dose-dependently. CONCLUSION: Based on these results, P. eryngii protein has a potential application in functional foods as a natural anti-tumour agent with immunostimulatory activity.

Concentrations and health risks of lead, cadmium, arsenic, and mercury in rice and edible mushrooms in China.

In this study, four common heavy metals, lead (Pb), cadmium (Cd), arsenic (As) and mercury (Hg) in rice and edible mushrooms of China were studied to evaluate contamination level and edible safety. Ninety two (92) rice samples were collected from the main rice growing regions in China, and 38 fresh and 21 dry edible mushroom samples were collected from typical markets in Nanjing City. The analyzed metal concentrations were significantly different between rice and edible mushroom samples (p<0.05). The results showed that Pb, Cd and As contents in 4.3%, 3.3% and 2.2% rice samples respectively, were above maximum allowable concentration (MAC). In fresh edible mushroom, Pb and Hg contents in 2.6% samples were above MAC, respectively. However, only Hg content in 4.8% dry edible mushroom samples was above its MAC. Therefore, more than 95% rice and edible mushroom samples in our test had high edible safety.