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.

Effect of ultrasonic-ionic liquid pretreatment on the hydrolysis degree and antigenicity of enzymatic hydrolysates from whey protein.

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins.

Small Molecule Oligopeptides Isolated from Walnut (Juglans regia L.) and Their Anti-Fatigue Effects in Mice.

Walnut (Juglans regia L.) is unique for its extensive biological activities and pharmaceutical properties. There are few studies on walnut oligopeptides (WOPs), which are small molecule peptides extracted from walnuts. This study aimed to evaluate the anti-fatigue effects of WOPs on ICR mice and explore the possible underlying mechanism. Mice were randomly divided into four experimental sets and each set of mice were then randomly divided into four groups. The vehicle group was administered distilled water, and the three WOP intervention groups were orally administered WOP solution at a dose of 110, 220, and 440 mg/kg of body weight, respectively. After 30 days of WOP intervention, the anti-fatigue activity of WOPs were evaluated using the weight-loaded swimming test and by measuring the change of biochemical parameters, glycogen storage and energy metabolism enzymes, anti-oxidative capacity and mitochondrial function. It was observed that WOPs could significantly prolong the swimming time, decrease the accumulation of lactate dehydrogenase (LDH), creatine kinase (CK), blood urea nitrogen (BUN) and blood lactic acid (BLA), and increased the glycogen storage of liver and gastrocnemius muscle. WOPs also markedly inhibited fatigue induced oxidative stress by increasing the activity of superoxide dismutase (SOD), glutathione peroxidase (GPX) and decreasing the content malondialdehyde (MDA). Notably, WOPs improved the activity of pyruvate kinase (PK), succinate dehydrogenase (SDH), Na+-K+-ATPase, and enhanced the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content in skeletal muscles of mice. These results suggest that WOPs have beneficial anti-fatigue effects, which may be attributed to their positive effects on increasing glycogen storage, improving energy metabolism, inhibiting oxidative stress, enhancing mitochondrial function in skeletal muscle, and ameliorating the cell damage and the muscular injury.

Ginseng (Panax ginseng Meyer) Oligopeptides Protect Against Binge Drinking-Induced Liver Damage through Inhibiting Oxidative Stress and Inflammation in Rats.

Panax ginseng C.A. Meyer (ginseng) is an edible and traditional medicinal herb, which is reported to have a wide range of biological activity and pharmaceutical properties. There were more studies on ginsenoside and polysaccharides, but fewer on ginseng oligopeptides (GOPs), which are small molecule oligopeptides extracted from ginseng. The present study was designed to investigate the effects and underlying mechanism of ginseng oligopeptide (GOPs) on binge drinking-induced alcohol damage in rats. Sprague Dawley rats were randomly assigned to six groups (n = 10), rats in normal control group and alcohol model group was administered distilled water; rats in four GOPs intervention groups (at a dose of 0.0625, 0.125, 0.25, 0.5 g/kg of body weight, respectively) were administered GOPs once a day for 30 days. Experiment rats were intragastrically administered ethanol at a one-time dose of 7 g/kg of body weight after 30 days. The liver injury was measured through traditional liver enzymes, inflammatory cytokines, expression of oxidative stress markers, and histopathological examination. We found that the GOPs treatment could significantly improve serum alanine aminotransferase and aspartate aminotransferase, plasma lipopolysaccharide, and inflammatory cytokine levels, as well as the oxidative stress markers that were altered by alcohol. Moreover, GOPs treatment inhibited the protein expression of toll-like receptor 4, and repressed the inhibitor kappa Bα and nuclear factor-κB p65 in the liver. These findings suggested that GOPs have a significant protective effect on binge drinking-induced liver injury, and the mechanism possibly mediated by the partial inhibition of lipopolysaccharide-toll-like receptor 4-nuclear factor-κB p65 signaling in the liver.

Ginseng oligopeptides protect against irradiation-induced immune dysfunction and intestinal injury.

Intestinal injury and immune dysfunction are commonly encountered after irradiation therapy. While the curative abilities of ginseng root have been reported in prior studies, there is little known regarding its role in immunoregulation of intestinal repairability in cancer patients treated with irradiation. Our current study aims to closely examine the protective effects of ginseng-derived small molecule oligopeptides (Panax ginseng C. A. Mey.) (GOP) against irradiation-induced immune dysfunction and subsequent intestinal injury, using in vitro and in vivo models. Expectedly, irradiation treatment resulted in increased intestinal permeability along with mucosal injury in both Caco-2 cells and mice, probably due to disruption of the intestinal epithelial barrier, leading to high plasma lipopolysaccharide (LPS) and pro-inflammatory cytokines levels. However, when the cells were treated with GOP, this led to diminished concentration of plasma LPS and cytokines (IL-1 and TNF-α), suggesting its dampening effect on inflammatory and oxidative stress, and potential role in restoring normal baseline intestinal permeability. Moreover, the Caco-2 cells treated with GOP showed high trans-epithelial electrical resistance (TEER) and low FITC-dextran paracellular permeability when compared to the control group. This could be explained by the higher levels of tight junction proteins (ZO-1 and Occludin) expression along with reduced expression of the apoptosis-related proteins (Bax and Caspase-3) noticed in the GOP-treated cells, highlighting its role in preserving intestinal permeability, through prevention of their degradation while maintaining normal levels of expression. Further confirmatory in vivo data showed that GOP-treated mice exhibited high concentrations of lymphocytes (CD3+, CD4+, CD8+) in the intestine, to rescue the irradiation-induced damage and restore baseline intestinal integrity. Therefore, we propose that GOP can be used as an adjuvant therapy to attenuate irradiation-induced immune dysfunction and intestinal injury in cancer patients.

Ginseng (Panax ginseng Meyer) oligopeptides regulate innate and adaptive immune responses in mice via increased macrophage phagocytosis capacity, NK cell activity and Th cells secretion.

Traditionally used as a restorative medicine, ginseng (Panax ginseng Meyer) has been the most widely used and acclaimed herb in Chinese communities for thousands of years. To investigate the immune-modulating activity of ginseng oligopeptides (GOP), 420 healthy female BALB/c mice were intragastrically administered distilled water (control), whey protein (0.15 g per kg body weight (BW)), and GOP 0.0375, 0.075, 0.15, 0.3 and 0.6 g per kg BW for 30 days. Blood samples from mice were collected from the ophthalmic venous plexus and then sacrificed by cervical dislocation. Seven assays were conducted to determine the immunomodulatory effects of GOP on innate and adaptive immune responses, followed by flow cytometry to investigate spleen T lymphocyte sub-populations, multiplex sandwich immunoassays to investigate serum cytokine and immunoglobulin levels, and ELISA to investigate intestinally secreted immunoglobulin to study the mechanism of GOP affecting the immune system. Our results showed that GOP was able to enhance innate and adaptive immune responses in mice by improving cell-mediated and humoral immunity, macrophage phagocytosis capacity and NK cell activity. Notably, the use of GOP revealed a better immune-modulating activity compared to whey protein. We conclude that the immune-modulating activity might be due to the increased macrophage phagocytosis capacity and NK cell activity, and the enhancement of T and Th cells, as well as IL-2, IL-6 and IL-12 secretion and IgA, IgG1 and IgG2b production. These results indicate that GOP could be considered a good candidate that may improve immune functions if used as a dietary supplement, with a dosage that ranges from 0.3 to 0.6 g per kg BW.

Suppression of TNF-α and free radicals reduces systematic inflammatory and metabolic disorders: Radioprotective effects of ginseng oligopeptides on intestinal barrier function and antioxidant defense.

Irradiation therapy is markedly associated with intestinal injure and oxidant stress. This study aimed to investigate the effects of ginseng (Panax ginseng C.A. Mey.) oligopeptides (GOP) on irradiation-induced intestinal injury and antioxidant defense in mice. BALB/c mice (8 weeks old) were randomly divided into six groups: vehicle control, irradiation control (IR), IR+whey protein [0.30 g/kg body weight (BW)], IR+GOP 0.15 g/kg BW, IR+GOP 0.30 g/kg BW and IR+GOP 0.60 g/kg BW. Postirradiation 30-day survival trial, white blood cells count and bone marrow hematopoietic system damage were performed to identify the injury degree induced by irradiation. Then, histopathology analysis was observed and intestinal permeability in vivo was quantified with fluorescein isothiocyanate-dextran. The enzyme-linked immunosorbent assay was used to determine antioxidant ability, plasma inflammatory cytokines, diamine oxidase (DAO) and endotoxin (LPS) levels. The immunohistochemistry assay was used to analyze the expression levels of tight junction proteins. We found that GOP-treated mice exhibited lower concentrations of plasma LPS and DAO and decreased instructors of inflammatory and oxidative stress which were linked to the lower intestinal permeability and higher tight junction proteins expression. The blockage of GOP was linked with the reduction of TNF-α and free radicals. The 15-day pretreatment of GOP could exhibit radioprotective effects, and another 15-day posttreatment benefited the quick repair of irradiation-induced injury. We confirm that GOP would exhibit effective therapeutic value on attenuating irradiation-induced hematopoietic, gastrointestinal and oxidative injury in cancer patients.

Sea cucumber (Codonopsis pilosula) oligopeptides: immunomodulatory effects based on stimulating Th cells, cytokine secretion and antibody production.

This study aimed to investigate the immunomodulating activity of small molecule oligopeptides from sea cucumber (Codonopsis pilosula) (SOP) in mice. Seven assays were performed to determine the immunomodulatory effects, including splenic lymphocyte proliferation and delayed-type hypersensitivity assays (cell-mediated immunity), IgM antibody response of spleen to sheep red blood cells (SRBC) and serum hemolysin level assays (humoral immunity), the carbon clearance assay and the phagocytic capacity of peritoneal cavity phagocytes assay (macrophage phagocytosis), and the NK cell activity assay. Spleen T lymphocyte subpopulations, multiplex sandwich immunoassays of serum cytokine and immunoglobulin levels and enzyme-linked immunosorbent assays for small intestinal secretory immunoglobulin were performed to study the mechanism by which SOP affects the immune system. We found that SOP could improve immune functions in mice, which may be due to the enhancement of the functions of cell-mediated immunity, humoral immunity, macrophage phagocytosis and NK cell activity. From the cellular and molecular assays, we postulated that the immunomodulatory effects are most likely attributed to the stimulation of Th cells, cytokine secretion and antibody production.

Bacterial Diversity Analysis during the Fermentation Processing of Traditional Chinese Yellow Rice Wine Revealed by 16S rDNA 454 Pyrosequencing.

Rice wine is a traditional Chinese fermented alcohol drink. Spontaneous fermentation with the use of the Chinese starter and wheat Qu lead to the growth of various microorganisms during the complete brewing process. It's of great importance to fully understand the composition of bacteria diversity in rice wine in order to improve the quality and solve safety problems. In this study, a more comprehensive bacterial description was shown with the use of bacteria diversity analysis, which enabled us to have a better understanding. Rarefaction, rank abundance, alpha Diversity, beta diversity and principal coordinates analysis simplified their complex bacteria components and provide us theoretical foundation for further investigation. It has been found bacteria diversity is more abundant at mid-term and later stage of brewing process. Bacteria community analysis reveals there is a potential safety hazard existing in the fermentation, since most of the sequence reads are assigned to Enterobacter (7900 at most) and Pantoea (7336 at most), followed by Staphylococcus (2796 at most) and Pseudomonas (1681 at most). Lactic acid bacteria are rare throughout the fermentation process which is not in accordance with other reports. This work may offer us an opportunity to investigate micro ecological fermentation system in food industry.

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.

Optimization of culture medium compositions for gellan gum production by a halobacterium Sphingomonas paucimobilis.

The effect of culture medium compositions on gellan gum production produced by fermentation with a halobacterium Sphingomonas paucimobilis QHZJUJW CGMCC2428 was studied. In this work, a fractional factorial design was applied to investigate the main factors that affected gellan gum production by S. paucimobilis QHZJUJW CGMCC2428. Sucrose was the best carbon source for gellan gum and peptone displayed better inducing effect. Central composite design and response surface methodology were adopted to derive a statistical model for optimizing submerged culture medium composition. These experimental results showed that the optimum culture medium for producing gellan gum was composed of 40.00 (w/v) sucrose, 3.00% peptone (w/v), MgSO4 (w/v), 9.20% KH2PO4 (w/v), 7.50% Na2HPO4 (w/v), 4.30% K2SO4 (w/v), pH 6.8-7.0. The maximal gellan gum was 19.89±0.68 g/L, which was agreed closely with the predicated value (20.12 g/L). After incubated for 72 h under the optimized culture medium in 5-L bioreactor, the gellan gum fermentation reached about 19.90±0.68 g/L, which was higher than that in the initial cultivation medium.

Applications and mechanisms of ionic liquids in whole-cell biotransformation.

Ionic liquids (ILs), entirely composed of cations and anions, are liquid solvents at room temperature. They are interesting due to their low vapor pressure, high polarity and thermostability, and also for the possibility to fine-tune their physicochemical properties through modification of the chemical structures of their cations or anions. In recent years, ILs have been widely used in biotechnological fields involving whole-cell biotransformations of biodiesel or biomass, and organic compound synthesis with cells. Research studies in these fields have increased from the past decades and compared to the typical solvents, ILs are the most promising alternative solvents for cell biotransformations. However, there are increasing limitations and new challenges in whole-cell biotransformations with ILs. There is little understanding of the mechanisms of ILs' interactions with cells, and much remains to be clarified. Further investigations are required to overcome the drawbacks of their applications and to broaden their application spectrum. This work mainly reviews the applications of ILs in whole-cell biotransformations, and the possible mechanisms of ILs in microbial cell biotransformation are proposed and discussed.

Production and identification of mannosylerythritol lipid-A homologs from the ustilaginomycetous yeast Pseudozyma aphidis ZJUDM34.

Mannosylerythritol lipids (MELs) are mainly produced by strains of the genus Pseudozyma and by Ustilago maydis. These glycolipid biosurfactants exhibit not only excellent surface-active properties but also versatile bioactivities. Mannosylerythritol lipid-A (MEL-A) is worth investigating due to its self-assembling property. In this work, crude MELs were produced by resting Pseudozyma aphidis ZJUDM34 cells using different culture media. MEL-A fractions were isolated and identified using high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) and gas chromatography combined with mass spectrometry (GC-MS). The results showed that MEL-A homologs had long unsaturated fatty acid chains, and the chain lengths range from C8 to C20. Nuclear magnetic resonance (NMR) was employed to confirm the chemical structures of the MEL-A homologs. Fermentation medium without NaNO3 and medium with manganese ions enhanced MEL-A production by Pseudozyma aphidis ZJUDM34.

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens, Phlebia radiata.

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg(-1)). Combined with the data of laccase and manganese peroxidase, oxalate and H2O2 were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6-114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H2O2 and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.

Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08.

Biotransformation of phytosterol (PS) by a newly isolated mutant Mycobacterium neoaurum ZJUVN-08 to produce androstenedione has been investigated in this paper. The parameters of the biotransformation process were optimized using fractional factorial design and response surface methodology. Androstenedione was the sole product in the fermentation broth catalyzed by the mutant M. neoaurum ZJUVN-08 strain. Results showed that molar ratio of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to PS and substrate concentrations were the two most significant factors affecting androstenedione production. By analyzing the statistical model of three-dimensional surface plot, the optimal process conditions were observed at 0.1 g/L inducer, pH 7.0, molar ratio of HP-ß-CD to PS 1.92:1, 8.98 g/L PS, and at 120 h of incubation time. Under these conditions, the maximum androstenedione yield was 5.96 g/L and nearly the same with the non-optimized (5.99 g/L), while the maximum PS conversion rate was 94.69% which increased by 10.66% compared with the non-optimized (84.03%). The predicted optimum conditions from the mathematical model were in agreement with the verification experimental results. It is considered that response surface methodology was a powerful and efficient method to optimize the parameters of PS biotransformation process.

A novel one-step microbial transformation of betulin to betulinic acid catalysed by Cunninghamella blakesleeana.

Betulinic acid and its derivatives are potential bioactive compounds present in nature. This study investigated the biotransformation of betulin to betulinic acid by Cunninghamella blakesleeana cells. LC-MS analysis demonstrated that betulin could be transformed into at least five products from cultured C. blakesleeana cells, among which betulinic acid was the most important. The presented method provides an attractive alternative approach to chemical synthesis, because is less time-consuming and more environmentally friendly. C. blakesleeana can transform betulin into potent derivatives with high pharmacological activities.

The synergistic effect on production of lignin-modifying enzymes through submerged co-cultivation of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora using agricultural residues.

The lignin-modifying enzymes (LMEs) play an important role in decomposition of agricultural residues, which contain a certain amount of lignin. In this study, the production of LMEs by three co-cultivated combinations of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora and the respective monocultures was comparatively investigated. Laccase and manganese peroxidases (MnP) were significantly promoted in the co-culture of P. radiata and D. squalens, and corncob was verified to be beneficial for laccase and MnP production. Moreover, laccase production by co-culture of P. radiata and D. squalens with high ratio of glucose to nitrogen was higher than low ratio under carbon- and nitrogen-meager conditions. New laccase isoenzymes measured by Native-PAGE were stimulated by co-cultured P. radiata with D. squalens or C. subvermispora, respectively, growing in the defined medium containing corncob, but the expression of laccase was greatly restrained by the co-culturing of D. squalens with C. subvermispora. This study showed that the synergistic and depressing effects of co-cultivation of P. radiata, D. squalens and C. subvermispora on LMEs were species specific.

Preparative isolation and purification of xanthohumol from hops (Humulus lupulus L.) by high-speed counter-current chromatography.

Xanthohumol (XN) and related prenylflavonoids are the main bioactive components of hops (Humulus lupulus L.). The current work is to investigate the use of high-speed counter-current chromatography (HSCCC) in search for high isolation of xanthohumol from hops. A solvent system consisted of n-hexane-ethyl acetate-methanol-water at a volume ratio of 5:5:4:3 was employed. The results demonstrated that the constructed method could be well applied for the isolation of xanthohumol from hops extract. After HSCCC isolation procedure, the purity of xanthohumol was over 95% assayed by HPLC and the yield of extraction was 93.60%. The chemical structure identification of xanthohumol was carried out by UV, (1)H NMR and (13)C NMR. The present results demonstrated that xanthohumol could be efficiently obtained using a single HSCCC step from H. lupulus L. extract.

Production of 8-prenylnaringenin from isoxanthohumol through biotransformation by fungi cells.

8-Prenylnaringenin (8PN), which presents in hop, enjoys fame as the most potential phytoestrogen. Although a number of health effects are attributed to 8PN, few reports are available about the production of it. In this work, screening of fungi to efficiently transform isoxanthohumol (IXN) into 8PN was designed. The biotransformation of IXN was significantly observed in Eupenicillium javanicum, Cunninghamella blakesleana, and Ceriporiopsis subvermispora under five kinds of transformation conditions. As a comparative result of IXN transformation, E. javanicum was the optimal biocatalyst to produce 8PN. Transformation caused by growing precultured fungal mycelia, a process designated as G2, was a favorable condition for IXN transformation in view of the yield of 8PN. The possible transformation pathway of 8PN bioproduction is postulated in this work. The construction of fungus and transformation mode derived from the current work is viable and an alternative procedure for 8PN formation.