Bistage control of pH for improving exopolysaccharide production from mycelia of Ganoderma lucidum in an air-lift fermentor

It was found that pH control definitely affects mycelial cell growth and exopolysaccharide (EPS) production of the mycelial cultivation of Ganoderma lucidum. Compared to the case of uncontrolled pH cultivation, a culture system whose pH was kept constant at 3 and 6 exhibited improved mycelial cell growth and EPS production, respectively. The bistage pH control technique, that is, shifting the pH from 3 to 6 at the initial phase of the exponential growth, is introduced to improve cell growth and EPS production. This technique can greatly increase EPS production to 20.1 g/l from 4.1 g/l in the case of uncontrolled pH cultivation, without adverse effects on cell growth as in the case of constant maintenance of a high pH. It was also proved that bistage pH control retained the desirable morphologies of the mycelia during cultivation and resulted in low viscosity and yield stress of the culture broth. It will be useful for the application of the culture process to mycelial growth in a large-scale fermentor.     

Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining

Ganoderma lucidum is a famous fungus. The triterpenoids are the main bioactive components and exhibit various pharmacological activities. However, the scarcity of the pure triterpenoid has greatly restrained the modern research of G. lucidum. The present paper first describes a convenient method to separate the main triterpenoids from G. lucidum using counter-current chromatography (CCC) technique. Ganoderic acid C6 (38 mg), E (76 mg), F (32 mg) were successfully separated from the crude extract in 1 day with the HPLC purity of above 90% using stepwise CCC. Ganoderic acid G (36 mg), A (64 mg), B (25 mg), D (28 mg) and ganoderenic acid D (11 mg) were separated in 2 days with the HPLC purity of above 90% using a combination of stepwise CCC and pH-zone-refining CCC. The method presented in the paper is much quicker and easier than the previous methods.     

Effect of mycelial culture broth of Ganoderma lucidum on the growth characteristics of human cell lines

Two types of purified samples, water-soluble (sample A; M. W, 1.2×10⁶ dalton) and water-insoluble (sample C; M. W., 1.0×10⁶ dalton) samples, were obtained through consecutive separation processes from the culture broth of Ganoderma lucidia mycelium. It was found that both samples from the culture broth were very effective in inhibiting the growth of several human cancer cell lines, having a 93-85% growth inhibition on Hep3B, AGS and A549 with the least cytotoxicity on the normal human lung cell line, WRL68 of less than 25% the highest supplementation concentration of 1.0 mg/l. In general, the sample C showed greater inhibition of cancer cell growth than the sample A. The same trend was also observed in antimutagenicity using the Chinese hamster ovary cell line (CHO test) or Salmonella typhimurium (Ames test). The CHO test showed that sample C had higher antimutagenicity on mutagens 4NQO or MMNG than sample A (approximately 40% vs approximately 25%). The percentage of antimutagenicity from the Ames test was lower than that from the CHO test, possibly due to the difference in the sensitivity of mutagens. The water-insoluble sample greatly enhanced the growth of the human T cell line (H9) up to 1×10⁵ with sample supplementation at 1.0 mg/l concentration from 4.3×10⁴ without sample supplementation as well as improved the secretion level of both IL-6 and TNF-α up to 100 pg/ml from approximately 40 pg/ml without sample supplementation. The kinetics of response to the immune cell growth was illustrated by the response time obtained when the sample concentration was increased. The water-insoluble sample can be used for effectively treating cancer in that it accelerated apoptosis of human carcinoma cells up to 70% compared to less than 50% for the control. The sample also increased the differentiation ratio of HL-60 cells up to 58% after four days of cultivation, compared to 18% in the case of no sample supplementation. These results can be used in implying that the insoluble part of G. lucidium mycelium culture broth must be related to controlling signal transduction, resulting in the regulation of cancer cell growth.     

Quantification of total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum by near infrared spectroscopy and chemometrics

Two chemometrics, the partial least-squares (PLS) and radial basis function (RBF) network were performed to develop a quantification method for total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum from different origins based on near infrared reflectance spectroscopy (NIR). The influences of spectral window and spectral pre-treatments were initially studied in the construction of PLS model. The best result was obtained when the standard normal transformation (SNV) +1st derivative spectrum over 4100–7750 cm⁻¹ was used for the modelling. Then based on each principle, both of the two models were optimised respectively. The final results with high determination coefficient (R²) (higher than 0.973, 0.989 for PLS and RBF, respectively) and low root mean square errors of prediction (RMSEP) (low to 0.1109 and 0.01298 for polysaccharides and triterpenoids, respectively) confirm the good predictability of the two models. The overall results show that NIR spectroscopy combined with chemometrics can be efficiently utilised for accurate analysis of routine chemical compositions in G. lucidum and G. atrum.     

Changes in some components of soymilk during fermentation with the basidiomycete Ganoderma lucidum

Soymilk was fermented with the basidiomycete Ganoderma lucidum WZ02 and the changes in the contents of polysaccharide, sugars, crude protein, B-vitamins, free amino acids and isoflavones were analyzed. Polysaccharide and crude protein were increased by the fermentation of G. lucidum while most free amino acids were reduced. The flatulence factor (e.g. stachyose and raffinose) was significantly decreased and stachyose was not detected after 72 h of fermentation. The contents of thiamin, riboflavin, and niacin were increased during the fermentation. Most of isoflavone glycosides were converted to aglycones and the contents of daidzein and genistein were increased by the fermentation of G. lucidum. The results suggested that fermentation by G. lucidum could improve the acceptability and health properties of soymilk.     

Bioactive dammarane-type triterpenoids derived from the acid hydrolysate of Gynostemma pentaphyllum saponins

From an acid hydrolysate of the crude saponins of Gynostemma pentaphyllum, three triterpenes, including a new compound (23S)-3β-hydroxydammar-20,24-dien-21-oic acid 21,23-lactone (1) and two known aglycons (20S, 23R)-3β,20β-dihydroxydamma-24-dien-21-oic acid 21,23-lactone (2) and (20S, 24S)-20,24-epoxydammarane-3β,12β,25-triol (3), were isolated. Their structures were established on the basis of extensive spectral evidence (HR-ESI-MS, IR, 1D and 2D-NMR experiments). In bioactive assays in vitro, compound 1 was found to have potent cytotoxicity against the human breast cancer cells MDA-MB-435, whereas compounds 2 and 3 exhibited modest inhibitory activity toward porcine pancreatic lipase. The results indicated that acid treatment of G. pentaphyllum extract could produce diverse structures with interesting bioactivity.     

Effect of fermentation time on antioxidative activities of Ganoderma lucidum broth using leguminous plants as part of the liquid fermentation medium

Oxidative damage plays an important role in the pathology of human diseases. Ganoderma lucidum, a medicinal fungus, has been used for thousands of years in traditional Oriental medicine. It is reported to have antioxidant functions such as inhibition of lipid peroxidation. The objective of the present study was to investigate the effect of fermentation time on the antioxidative activities of G. lucidum broth filtrate using leguminous plants as part of the liquid fermentation medium. Inhibition of Cu²⁺-induced oxidation of human low-density lipoprotein (LDL), DPPH radical-scavenging activity, total phenolic compounds, isoflavones and protocatechuic acid were measured to evaluate the antioxidant activity of G. lucidum fermentation broth filtrate. Our results showed that black soybean and Astragalus membranaceus improved the antioxidant activity of the G. lucidum fermentation broth filtrate. Protocatechuic acid was identified by LC–MS as the antioxidant compounds whose relative potency of inhibiting LDL oxidation to Trolox is 1.55. Protocatechuic acid showed positive correlation with the antioxidant activity of the fermentation broth filtrate while isoflavones did not contribute to antioxidant activity.     

Biotransformation of ginsenoside Rd in the ginseng extraction residue by fermentation with lingzhi (Ganoderma lucidum)

Ginseng and lingzhi (Ganoderma lucidum) both are valuable traditional Chinese medicines and have been extensively utilised in functional foods and traditional medicines in many Asian countries. However, massive quantity of ginseng residue is produced after extraction of ginseng which still contains a lot of bioactive compounds such as ginsenosides. The goal of this study was to reuse the American ginseng extraction residue as the fermentation medium of G. lucidum to produce bioactive ginsenoside enriched biotransformation products. The changes of ginsenosides in the fermentation products were analysed during fermentation. Our results showed that after 30 days of fermentation, ginsenoside Rg1, Rd, and compound K (CK) significantly increased, especially Rd, while other ginsenosides (Re, Rb1 and Rc) decreased during fermentation. Ginsenoside Rd is the major ginsenoside in the final fermentation product. Furthermore, the biotransformation of ginsenosides was the major reaction in this fermentation process.     

Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus

Eight strains of Lactobacillus plantarum, 6 strains of Lactobacillus casei, Lactobacillus acidophilus LA-5, and Bifidobacterium lactis BB-12 were screened for β-glucosidase activity. We then proceeded to investigate the enzymatic potential of selected strains for bioconversion of delphinidin and malvidin glycosides to their metabolites. L. plantarum and L. casei strains showed the highest cell-envelope associated β-glucosidase activity. Intracellular β-glucosidase activity from B. lactis BB-12 was up to 287-fold higher than that of the other strains. The L. acidophilus strain showed low β-glucosidase activity, both, intra and extracellularly. No aglycons were detected in bacterial extract reactions with anthocyanin glycosides. Delphinidin-3-glucoside underwent chemical degradation to form mainly gallic acid, although delphinidin-3-glucoside degradation due to B. lactis BB-12 and enzymatic activity towards chemically-formed metabolites due to L. casei LC-01 were observed. Incubation of malvidin-3-glucoside with B. lactis BB-12, L. plantarum IFPL722, and L. casei LC-01 cell-free extracts led to different patterns of gallic, homogentisic and syringic acid formation.     

Biochemical characterization of a proteoglycan complex from an edible mushroom Ganoderma lucidum fruiting bodies and its immunoregulatory activity

A fraction, LZ-D-7, was isolated from the fruiting body of the edible mushroom Ganoderma lucidum using a series of chromatographic steps. Biochemical experiments showed that the complex is a proteoglycan and has a carbohydrate percentage of 95.3%, along with a protein percentage of 3.3%. Methylation analysis showed the polysaccharide moiety of LZ-D-7 had a backbone of 1,4-disubsituted-glucopyranosyl and 1,2,6-trisubstituted-glucopyranosyl residues. The branches were mainly composed of 1-substituted-glucopyranosyl residue, which was attached to 1,2,6-trisubstituted-glucopyranosyl residue via 2 or 6 position. Investigation of modulation of LZ-D-7 on BALB/c mouse spleen lymphocytes cells resulted in a three to four-fold increase of MSLs percentage. Analysis of activation and proliferation of MSLs indicated that LZ-D-7 activating most of the activated cells were B cells. The B cells were enlarged, expressed CD⁷¹⁺ and CD²⁵⁺ on the cell surface.     

Antioxidant activities from the aerial parts of Platycodon grandiflorum

In order to obtain basic data necessary for the utilisation of aerial parts from Platycodon grandiflorum as a functional substance in Korea, the antioxidant activities of solvent fractions from the ethanol extract of P. grandiflorum aerial parts were examined. The butanol fraction from P. grandiflorum showed the most potent antioxidant activities in each assay, showing 91.31% in the DPPH radical scavenging method, 99.62% in the ABTS radical scavenging method, 7.84% in the reducing power method, and 1.29% in the FRAP method at a concentration of 10 mg/ml. The DPPH, ABTS, reducing power, and FRAP assay indicated that the butanol fraction of aerial parts of P. grandiflorum was the most potent radical-scavengers and reducing agents compared to the other two extracts. Therefore, our study verified that the butanol fraction has strong antioxidant activities which are correlated with its high level of phenolics, particularly luteolin-7-O-glucoside and apigenin-7-O-glucoside. This extract of P. grandiflorum aerial parts can be utilised as an effective and safe source of functional food materials such as natural antioxidants.     

Evaluation of in vivo antioxidant activities of Ganoderma lucidum polysaccharides in STZ-diabetic rats

Effect of Ganoderma lucidum polysaccharides treatment on blood glucose, serum insulin level, lipid peroxidation, nonenzymic and enzymic antioxidants in the plasma and liver of streptozotocin (STZ)-induced diabetic rats was studied. Adult male rats of Wistar strain, weighing 195 to 250 g, were randomized into control and experimental groups. Experiment group rats were induced diabetes by administration of STZ (45 mg/kg b.wt.) intraperitoneally. The diabetic rats were treated with G. lucidum polysaccharides (60, 120, 180 mg/kg b.wt.) dissolved in 15% dimethyl sulphoxide (DMSO) for 30 days. The normal control rats were treated with 15% DMSO for 30 days. Streptozotocin treatment elevated the levels of lipid peroxidation markers (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes), and reduced nonenzymic antioxidants (vitamin C and reduced glutathione, vitamin E) levels, and enzymic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) activities in the plasma and liver of untreated diabetic control rats. Decreased level of serum insulin and increased level of blood glucose (BG) were observed in the plasma of untreated diabetic control rats. G. lucidum polysaccharides treatment significantly and dose-dependently increased nonenzymic and enzymic antioxidants, serum insulin level and reduced lipid peroxidation, blood glucose levels in STZ-diabetic rats. From the present study, it can be concluded that G. lucidum polysaccharides can be considered as a potent antioxidant.     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.     

Protective effects of triterpenoids from Ganoderma resinaceum on H2O2-induced toxicity in HepG2 cells

Ganoderma resinaceum Boud. (Polyporeseae) has long been used for antioxidant, immunoregulation and liver protection. From the fruiting bodies of G. resinaceum, eight new lanostanoids, lucidones D–G (1–4), 7-oxo-ganoderic acid Z₂ (5), 7-oxo-ganoderic acid Z₃ (6), ganoderesin A (7), and ganoderesin B (8), together with six known lanostanoids (9–14) were isolated. The structures of new compounds were elucidated through extensive spectroscopic analysis. In an in vitro model, ganoderesin B (8), ganoderol B (10) and lucidone A (11) showed inhibitory effects against the increase of ALT and AST levels in HepG2 cells induced by H₂O₂ compared to a control group in the range of their maximum non-toxic concentration (MNTC). However, compounds 8, 10 and 11 displayed no anti-oxidant activities by DPPH assay. Meanwhile, activation for PXR (Pregnane X Receptor) of ganoderesin B (8), ganoderol B (10) and lucidone A (11) was evaluated; ganoderol (10) exhibited a vital activation for PXR-induced CYP3A4 expression. These results suggested that GTs (Ganoderma triterpenoids) exhibited hepatoprotective activities by lowering ALT and AST levels.     

Phenolic contents and antioxidant activities of bitter gourd (Momordica charantia L.) leaf, stem and fruit fraction extracts in vitro

Bitter gourd (Momordica charantia L.) has long been regarded as a food and medicinal plant. We investigated the antioxidant activity of the water extract of leaf, stem and fruit fractions by several in vitro systems of assay, namely DPPH radical-scavenging activity, hydroxyl radical-scavenging activity, β-carotene–linoleate bleaching assay, ferric reducing/antioxidant power (FRAP) assay and total antioxidant capacity. Total phenolic content was measured by Folin–Ciocalteu reagent. Identification of phenolic compounds was achieved using HPLC with the UV-diode array detection. The extracts of different fractions were found to have different levels of antioxidant activity in the systems tested. The leaf extract showed the highest value of antioxidant activity, based on DPPH radical-scavenging activity and ferric reducing power, while the green fruit extract showed the highest value of antioxidant activity, based on hydroxyl radical-scavenging activity, β-carotene–linoleate bleaching assay and total antioxidant capacity. The predominant phenolic compounds were gallic acid, followed by caffeic acid and catechin. The present study demonstrated that the water extract fractions of bitter gourd have different responses with different antioxidant methods. Total phenol content was shown to provide the highest association with FRAP assay in this present study (R² = 0.948).     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Effect of oxygen concentration in gas phase on sporulation and individual ganoderic acids accumulation in liquid static culture of Ganoderma lucidum

Effects of oxygen concentration within 21–100% in gaseous phase on the morphology and ganoderic acids (GAs) production by Ganoderma lucidum in liquid static culture were studied. A higher oxygen concentration increased individual GAs production, and more spores and higher total GA content were obtained at an oxygen level of 80%.     

Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin

β-Lactoglobulin A and β-lactoglobulin B were heated at 75°C in the absence and presence of α-lactalbumin, and the aggregation products were characterized by size exclusion chromatography in combination with multi-angle laser light scattering and electrophoretic techniques. α-Lactalbumin did not form aggregates when heated alone, but in admixture with β-lactoglobulin it was incorporated into both the disulphide-bonded and the hydrophobically associated aggregates as well as forming α-lactalbumin dimers and other oligomers. The presence of α-lactalbumin diminished the proportion of smaller aggregates and increased the number of very large aggregates within both variant protein mixtures. In the presence of α-lactalbumin, β-lactoglobulin A was converted into a series of disulphide-bonded and the hydrophobically associated aggregates more slowly, but with a greater proportion of hydrophobically associated aggregates, than β-lactoglobulin B. These patterns are similar to that when β-lactoglobulin A or B are heated on their own. These and other results indicate that the mechanism of aggregation of α-lactalbumin/β-lactoglobulin mixtures is governed by β-lactoglobulin.