Glycolysis characteristics of intracellular polysaccharides from Agaricus bitorquis (Quél.) sacc. Chaidam and its effects on intestinal flora from different altitudes of mice in vitro fermentation.

The glycolysis characteristics and effects on intestinal flora of polysaccharides from Agaricus bitorquis (Quél.) Sacc. Chaidam (ABIPs) in vitro fermentation by different altitudes of mice feces was examined, including low, medium, and high altitudes groups (LG, MG, and HG). In vitro, fermentation of ABIPs forty-eight hours resulted in a remarkable decrease in total sugar content and improvement of short-chain fatty acids (SCFAs) (mainly acetate, propionate, and butyrate), which simultaneously induced the composition of monose and uronic acids and SCFAs continuously change. Besides, ABIPs influenced the abundance and composition of the intestinal flora, generally increasing the abundance of probiotic bacteria (such as Bifidobacterium and Faecalibacterium) and decreasing the abundance of harmful bacteria (such as Phenylobacterium and Streptococcus) in all groups, with the highland biology core genus Blautia significantly enriched in LG and MG groups. It was also found that ABIPs enhanced pathways associated with biosynthesis and metabolism. In addition, correlation analysis speculated that the metabolism of SCFAs by ABIPs may be associated with genera such as Anaerostipes, Roseburia, and Weissella. ABIPs may protect organismal health by regulating hypoxic intestinal flora composition and metabolic function, and more superior fermentation performance was observed in MG compared to other groups.

Preparation and activity evaluation of angiotensin-I converting enzyme inhibitory peptides from protein hydrolysate of mulberry leaf.

Angiotensin-I converting enzyme (ACE) inhibitory peptides drew wide attention in the food industry because of their natural reliability, non-toxicity, and safety. However, the characteristics of ACE inhibitory peptides obtained from protein hydrolysate of mulberry leaf prepared by Flavourzyme were still unclear. Based on the single-factor test, the Plackett-Burman test and response surface test were used to determine the key factors affecting the ACE inhibition rate in mulberry leaf protein hydrolysate and the optimum conditions of enzymatic hydrolysis. The results showed that the optimum technical parameters were as follows: the ratio of material to liquid is 1: 25 (w / v, g/mL), the Flavourzyme to substrate ratio was 3,000 U/g, the temperature of enzymatic hydrolysis was 50°C, pH was 6.3, and the time of enzymatic hydrolysis was 2.9 h. The ACE inhibitory peptides in the mulberry leaf protein hydrolysates were purified by ultrafiltration and gel filtration, aiming to obtain the highest active component. The 12 peptide sequences were identified by reverse liquid chromatography-mass spectrometry, and then, they were docked to the crystal structure of human angiotensin-I converting enzyme (1O8A), and the interaction mechanisms of 12 peptide sequences and 1O8A were analyzed. The docking results showed that among the 12 peptide sequences, ERFNVE (792.37 Da), TELVLK (351.72 Da), MELVLK (366.72 Da), and FDDKLD (376.67 Da), all had the lowest docking energy, and inhibition constant. The chemosynthetic ERFNVE (IC50: 2.65 mg/mL), TELVLK (IC50: 0.98 mg/mL), MELVLK (IC50:1.90 mg/mL) and FDDKLD (IC50:0.70 mg/mL) demonstrated high ACE-inhibitory activity with competitive inhibition mode. These results indicated that the ACE-inhibiting peptides from mulberry leaf protein hydrolyzed (FHMP) had the potential activities to inhibit ACE and could be used as functional food or drugs to inhibit ACE. This work provides positive support for mining the biological activity of mulberry leaves in the treatment of hypertension.

Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

Phenolic Acids-Rich Fractions from Agaricus bitorguis (Quél.) Sacc. Chaidam ZJU-CDMA-12 Mycelia Modulate Hypoxic Stress on Hypoxia-Damaged PC12 Cells.

Hypoxia is a common pathological process in various clinical diseases. However, there is still a lack of effective anti-hypoxia active substances. Agaricus bitorguis (Quél.) Sacc Chaidam (ABSC) is a rare wild edible macrofungus that grows underground at high altitudes. Herein, intracellular phenolic acids-rich fractions (IPA) were extracted from ABSC ZJU-CDMA-12, and the structural characterization and anti-hypoxia activity of IPA on PC12 cells were elucidated as well. The results of HPLC-Q-TOF-MS illustrated that five kinds of IPA were isolated from ABSC, including (-)-epicatechin gallate, arabelline, yunnaneic acid D, 2'-O-p-hydroxybenzoyl-6'-O-trans-caffeoylgardoside,4'-O-methylgallocatechin-(4->8)-4'-O-methylepigallocatechin. IPA extracted from ABSC proved to show anti-hypoxia activity on hypoxia-damaged PC12 cells. Hypoxia enhanced reactive oxygen species (ROS) generation and reduced the mitochondrial membrane potential (ΔΨm) in PC12 cells, resulting in the inhibition of survival and induction of apoptosis in PC12 cells. Measurements of 100 µg/mL and 250 µg/mL IPA could significantly reduce hypoxia-induced damage in PC12 cells by decreasing overproduced intracellular ROS, improving ΔΨm, and reducing cell apoptosis rate. Our findings indicated that the IPA from ABSC potentially could be used as novel bioactive components applied to anti-hypoxia functional foods or medicines.

Ultrasound enhanced production of mycelia and exopolysaccharide by Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC), is a kind of rare edible macrofungi with a variety of biological ingredients, especially its polysaccharides. However, the low yield limits the popularity and promotion of rare edible macrofungi as well as its macrofungi polysaccharides. Hence, developing a positive and effective cultivation method is of great importance. Herein, an efficient ultrasonic (US) stimulation strategy was developed to improve mycelial growth and exopolysaccharides (EPS) biosynthesis from ABSC in submerged cultivation without light. A time design was employed to illustrate the effect of various process parameters including duration, starting point and times of US irradiation on ABSC productivity. 5 min US treatment for once upon ABSC after fermentation for 48 h could significantly improve EPS production and mycelia growth by above 26% and 15.03%, respectively. Furthermore, six times of 5 min US treatment could make the amount of EPS reach 218.78 ± 17.09 mg/g, which was 2.52-fold higher than that of the control. Moreover, the enhanced effect induced by US was further expounded by fermentation kinetics. Besides, the US treatment could increase mycelia permeability, change structure and reduce mycelial diameter to promote mass transfer, resulting in the improvement of EPS production and mycelia accumulation. The results demonstrated that the present proposed US intensification approach could be useful to boost up the fermentation of ABSC, which possibly applied to yield increase and fermentation product acquisition of macrofungi.

Chemical characterization, antioxidant properties and anticancer activity of exopolysaccharides from Floccularia luteovirens.

Two polysaccharides, ALF1 and ALF2 were obtained from the fermentation liquid of Floccularia luteovirens. These fractions had good performance in scavenging radicals and ALF1 exhibited obvious antioxidant activities. Further, linkage analysis and NMR were used to characterize the structures of ALF1. Linkage and NMR data comprehensively showed that ALF1 mainly contained six kinds of linkage type units as →4)-ß-D-Manp→, 1,3-α-Fucp→, α-L-Araf-C1→, →6)-ß-D-Galp-C1→, →4)-α-D-GlcAp-(1→ and →3)-ß-D-Glcp(1→. In addition, ALF1 had good bioactivities such as anticancer and antioxidant activities. ALF1 was proven to be able to inhibit tumor cells without affecting the normal cells. Besides, ALF1 improved the activities of SOD, GSH-Px and CAT, and decreased the production of MDA which result in protecting PC12 cells against H2O2-induced oxidative stress. ALF1 decreased ROS production, and stabilize mitochondrial membrane potential. The findings indicated that the fermentation liquid of Floccularia luteovirens could be used as a potential natural source of antioxidant.

Characterization and antioxidant activities of intracellular polysaccharides from Agaricus bitorquis (QuéL.) Sacc. Chaidam ZJU-CDMA-12.

The antioxidant activities of polysaccharides from the fruiting body (PFB), extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) from Agaricus bitorquis (QuéL.) Sacc. Chaidam ZJU-CDMA-12 in vitro were compared. IPS showed stronger antioxidant activities than PFB and EPS in vitro. Further purification and structure analyses indicated that IPS mainly consisted of three fractions (IPS-I, IPS-II and IPS-III). FT-IR and NMR data indicated that IPS was mainly composed of (1 â†’ 6)-linked α-d-glucose. There are significant differences of antioxidant activities among IPS-I, IPS-II and IPS-III fractions in vitro, and IPS-III showed stronger antioxidant activity than IPS-I and IPS-II. IPS-III also possesses a potent antioxidant ability inside HepG2 cells, and it could protect HepG2 cells from H2O2-induced cytotoxicity by scavenging overproduced cellular ROS and inhibiting SOD, CAT and GSH depletion to weaken lipid peroxidation. These findings suggested that IPS-III could be a novel antioxidant and that it could afford protection against H2O2-induced cytotoxicity and oxidative stress.

Polysaccharides from the Edible Mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam Show Anti-hypoxia Activities in Pulmonary Artery Smooth Muscle Cells.

Three kinds of new water-soluble polysaccharides (FA, FB and FC) were isolated from wild mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam by the classical method "water extraction and alcohol precipitation" and purified by column chromatography. The Mw of FA, FB and FC ranged from 5690 Da to 38,340 Da. The three polysaccharide fractions in the fruiting body were mainly composed of 4 kinds of monosaccharides, including glucose, galactose, mannose, and arabinose, among which glucose and galactose were the major monosaccharides. The FTIR and NMR spectroscopy indicated that the skeleton of three fractions composed of a (1→4)-α-D-glycosidic backbone containing α-D-mannopyranose. In vitro anti-hypoxia activity data showed that three polysaccharide fractions possessed a significant effect on inhibiting PASM cells apoptosis under hypoxia. Among them, FC at the concentration of 200 µg/mL revealed a significant anti-hypoxia effect. These results revealed that the intracellular polysaccharides possessed potent anti-hypoxic activity, which might be related to inhibiting LDH and NADPH oxidase expression and promoting the formation of 5-hydroxytryptamine, dopamine, endothelins, acetylcholine. More importantly, FC showed good performance inducing KV1.5 expression and prohibiting KIR6.2 formation at protein level.

Fungal chitosan from Agaricus bisporus (Lange) Sing. Chaidam increased the stability and antioxidant activity of liposomes modified with biosurfactants and loading betulinic acid.

Agaricus bisporus (Lange) Sing. Chaidam, a special brown mushroom with thick body indigenous in Chaidam basin, was used for fungal chitosan extraction. FTIR, XRD and DSC spectra showed that fungal chitosan was similar to commercial chitosan from aquatic sources. Fungal chitosan and commercial chitosan were used to coat on betulinic acid-loaded liposomes modified with biosurfactants mannosylerythritol lipid A (MEL-A), respectively. After chitosan coating, the mean size, zeta potential and encapsulation efficiency of both liposomes increased. The liposomes coated with fungal chitosan were discovered to have smaller size and higher zeta potential. Furthermore, the wall material MEL-A and coating material chitosan endue liposomes with increased antioxidant capacity. Fungal chitosan coated liposomes also have stronger antioxidant effects than commercial chitosan. The findings implied that the fungal chitosan coated liposomes modified with MEL-A can be considered as a promising delivery system with enhanced antioxidant effects for bioactive compounds.

Preparation, Characterization, and Antibacterial Effects of Chitosan Nanoparticles Embedded with Essential Oils Synthesized in an Ionic Liquid Containing System.

Several chitosan sodium tripolyphosphate (TPP) nanoparticles embedded with Torreya grandis aril essential oils (TEOs) were synthesized using an emulsion-ionic gelation technique. Mannosylerythritol lipid A (MEL-A), a type of biosurfactant, was selected as the emulsifier. In order to replace acetic acid, an ionic liquid (IL) was employed to dissolve chitosan. The physical properties, diameters, morphology, embedding rate, and antibacterial effects of those essential oil loaded chitosan (CS) nanoparticles were characterized. The results demonstrated that chitosan nanoparticles can be successfully prepared in an ionic liquid containing system and the diameters for nanoparticles in acetic acid and ionic liquid solutions are 144.1 ± 1.457 and 219.0 ± 4.045 nm. After loading with essential oils, the size increased to 349.6 ± 10.55 and 542.9 ± 16.74 nm, respectively. Antibacterial properties were investigated by the observation of the inhibition zone against S. aureus. The results revealed that TEO loaded nanoparticles synthesized in acid and IL aqueous systems have stronger antibacterial activities than CS nanoparticles.

Optimization of EPS Production and Characterization by a Halophilic Bacterium, Kocuria rosea ZJUQH from Chaka Salt Lake with Response Surface Methodology.

With the rising awareness of microbial exopolysaccharides (EPSs) application in various fields, halophilic microorganisms which produce EPSs have received broad attention. A newly identified Kocuria rosea ZJUQH CCTCC M2016754 was determined to be a moderate halobacterium on account of its successful adaption to the environment containing 10% NaCl. The optimal combination of fermentation medium compositions on EPS production was studied. In this work, a fractional factorial design was adopted to investigate the significant factors that affected EPS production. The factors of KCl and MgSO4 were found to have a profound impact on EPS production. We utilized central composite design and response surface methodology to derive a statistical model for optimizing the submerged culture medium composition. Judging from these experimental results, the optimum culture medium for producing EPSs was composed of 0.50% casein hydrolysate, 1.00% sodium citrate, 0.30% yeast extract, 0.50% KCl, 0.50% peptone, and 5.80% MgSO4 (initial pH 7.0). The maximal EPS was 48.01 g/L, which is close to the predicted value (50.39 g/L). In the validation experiment, the highest concentration of 70.64 g/L EPSs was obtained after 120 h under the optimized culture medium in a 5-L bioreactor. EPS from this bacterium was also characterized by differential scanning calorimetry (DSC) and Fourier transform infrared analysis (FT-IR). The findings in this study imply that Kocuria rosea ZJUQH has great potential to be exploited as a source of EPSs utilized in food, the pharmaceutical and agriculture industry, and in the biotreatment of hypersaline environments.

In Vitro Antioxidant Activities and in Vivo Anti-Hypoxic Activity of the Edible Mushroom Agaricus bisporus (Lange) Sing. Chaidam.

With the rising awareness of a healthy lifestyle, natural functional foods have gained much interest as promising alternatives to synthetic functional drugs. Recently, wild Agaricus bisporus (Lange) Sing. Chaidam has been found and artificially cultivated for its thick fresh body and excellent taste, with its antioxidant and anti-hypoxic abilities unknown. In this work, the antioxidant potential of its methanolic, 55% ethanolic, aqueous extracts and crude polysaccharide was evaluated in different systems. The results showed that polysaccharide was the most effective in scavenging ability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals, metal chelating activity and reducing power, with EC50 values of 0.02, 2.79, 1.29, and 1.82 mg/mL, respectively. Therefore, we further studied the anti-hypoxic activity of crude polysaccharide. The results turned out that polysaccharide (300 mg/kg) prolonged the survival time, decreased the blood urea nitrogen and lactic acid content as well as increased the liver glycogen significantly, compared with the blank control and the commercialized product Hongjingtian (p < 0.05). With such excellent activities, we purified the polysaccharide and analyzed its molecular weight (120 kDa) as well as monosaccharide components (glucose, fructose and mannose). This study indicated that wild Agaricus bisporus (Lange) Sing. Chaidam had strong potential to be exploited as an effective natural functional food to relieve oxidative and hypoxia stresses.