Production of bioactive metabolites by submerged fermentation of the medicinal mushroom Antrodia cinnamomea: recent advances and future development.

Edible and medicinal mushrooms have usually been considered as a sustainable source of unique bioactive metabolites, which are valued as promising provisions for human health. Antrodia cinnamomea is a unique edible and medicinal fungus widespread in Taiwan, which has attracted much attention in recent years for its high value in both scientific research and commercial applications owing to its potent therapeutic effects, especially for its hepatic protection and anticancer activity. Due to the scarcity of the fruiting bodies, the cultivation of A. cinnamomea by submerged fermentation appears to be a promising substitute which possesses some unique advantages, such as short culture time period and its high feasibility for scale-up production. However, the amount of fungal bioactive metabolites derived from the cultured mycelia of A. cinnamomea grown by submerged fermentation is much less than those obtained from the wild fruiting bodies. Hence, there is an urgent need to bridge such a discrepancy on bioactive metabolites between the wild fruiting bodies and the cultured mycelia. The objective of this article is to review recent advances and the future development of the mycelial submerged fermentation of A. cinnamomea in terms of enhancement for the production of fungal bioactive components by the optimization of culture conditions and the regulation of fungal metabolism. This review provides valuable information for further biotechnological applications of A. cinnamomea as well as other mushrooms being the source of bioactive ingredients by submerged fermentation.

Efficient Biosynthesis of Natural Yellow Pigments by Monascus purpureus in a Novel Integrated Fermentation System.

Because of the increasing demand for healthy and safe food, Monascus spp. have gained much attention as a sustainable source of natural food colorant. In this study, a novel integrated fermentation system consisting of surfactant and in situ extractant was established for efficiently producing yellow pigments by M. purpureus sjs-6. The maximum production of Monascus yellow pigment (669.2 U/mL) was obtained when 40% soybean oil (as extractant) was supplied at the beginning and 5 g/L Span-80 (as surfactant) was supplied at the 72nd h, which resulted in production 27.8-times of that of the control. Critical factors such as alleviating the product inhibition, increasing the membrane permeability, changing the hyphal morphology, and influencing the cell activity have been suggested as the underlying mechanisms. This system is of great significance for the bioprocess, which suffers product inhibition, and it can serve as a promising step for enhancing the yield of hydrophobic metabolites.

Using millet as substrate for efficient production of monacolin K by solid-state fermentation of Monascus ruber.

In this study, various grains such as rice, millet, corn, barley and wheat were used as raw materials for monacolin K production by solid-state fermentation of Monascus ruber. Among these substrates, millet was found to be the best one for monacolin K production, by which the yield reached 7.12 mg/g. For enhanced monacolin K production, the effects of fermentation time, charge amount, initial moisture content and inoculum volume were systematically investigated in the solid-state fermentation of M. ruber. Moreover, complementary carbon source and nitrogen source were added for further improving the production of monacolin K. Results showed that the maximum production of monacolin K (19.81 mg/g) could be obtained at the optimal conditions. Compared with the traditional red mold rice, using millet as substrate is promising for high production of monacolin K in the solid-state fermentation of M. ruber.

Current Advances on the Structure, Bioactivity, Synthesis, and Metabolic Regulation of Novel Ubiquinone Derivatives in the Edible and Medicinal Mushroom Antrodia cinnamomea.

In recent years, Antrodia cinnamomea has attracted great attention around the world as an extremely precious edible and medicinal mushroom. Ubiquinone derivatives, which are characteristic metabolites of A. cinnamomea, have shown great bioactivities. Some of them have been regarded as promising therapeutic agents and approved into clinical trial by the U.S. Food and Drug Administration. Although some excellent reviews have been published covering different aspects of A. cinnamomea, this review brings, for the first time, complete information about the structure, bioactivity, chemical synthesis, biosynthesis, and metabolic regulation of ubiquinone derivatives in A. cinnamomea. It not only advances our knowledge on the bioactive metabolites, especially the ubiquinone derivatives, in A. cinnamomea but also provides valuable information for the investigation on other edible and medicinal mushrooms.

Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology.

Natural yellow pigments produced by submerged fermentation of Monascus purpureus have potential economic value and application in the food industry. In the present study, the relationships among fermentation conditions (in terms of pH and shaking/agitation speed), mycelial morphology and the production of Monascus yellow pigments were investigated in both shake-flask and scale-up bioreactor experiments. In the shake-flask fermentation, the highest yield of the Monascus yellow pigments was obtained at pH 5.0 and a shaking speed of 180 rpm. Microscopic images revealed that these results were associated with the formation of freely dispersed small mycelial pellets with shorter, thicker and multi-branched hyphae. Further investigation indicated that the hyphal diameter was highly correlated with the biosynthesis of the Monascus yellow pigments. In a scaled-up fermentation experiment, the yield of yellow pigments (401 U) was obtained in a 200-L bioreactor, which is the highest yield to the best of our knowledge. The present findings can advance our knowledge on the conditions used for enhancing the production of Monascus yellow pigments in submerged fermentation and facilitate large-scale production of these natural pigments.

Induction of antroquinonol production by addition of hydrogen peroxide in the fermentation of Antrodia camphorata S-29.

BACKGROUND: Antroquinonol have significantly anti-tumour effects on various cancer cells. There is still lack of reports on regulation of environmental factors on antroquinonol production by Antrodia camphorata. RESULTS: An effective submerged fermentation method was employed to induce antroquinonol with adding H2 O2 . The production of antroquinonol was 57.81 mg L-1 after fermentation for 10 days when adding 25 mmol L-1 H2 O2 at day 4 of the fermentation process. Then, antroquinonol was further increased to 80.10 mg L-1 with cell productivity of 14.94 mg g-1 dry mycelium when the feeding rate of H2 O2 was adjusted to 0.2 mmol L-1 h-1 in the 7 L fermentation bioreactor. After inhibiting the generation of reactive oxygen species with the inhibitor diphenyleneiodoium, the synthesis of antroquinonol from A. camphorata was significantly reduced, and the yield was only 3.3 mg L-1 . CONCLUSION: The results demonstrated that addition of H2 O2 was a very effective strategy to induce and regulate the synthesis of antroquinonol in submerged fermentation. Reactive oxygen species generated by H2 O2 during fermentation caused oxidative stress, which induced the synthesis of antroquinonol and other chemical compounds. Moreover, it is very beneficial process to improve production and diversity of the active compounds during liquid fermentation of A. camphorata mycelium. © 2016 Society of Chemical Industry.

Composition of Unrecorded Distilled Alcohol (bai jiu) Produced in Small Rural Factories in Central China.

BACKGROUND: Unrecorded traditional distilled spirits (bai jiu, ) are made and used throughout rural China for everyday use and special occasions. Nearly every town or village has a distiller to supply the demand. In rural China, distilling bai jiu is legal and regulated lightly or not at all. The World Health Organization estimates that as much as 25% of all alcohol consumed in China is unrecorded alcohol, of which an unknown portion is unrecorded bai jiu. Little is known about the composition of unrecorded Chinese spirits from rural parts of the country. This study focused on white spirits because the high ethanol (EtOH) concentration makes them more likely to contribute to health risks compared to other types of lower alcohol by volume (ABV) Chinese unrecorded alcohol. METHODS: Researchers purchased samples of Chinese white spirits from small-factory distillers in central China. An independent laboratory conducted the analysis. Alcohol strength (ABV) was determined by hydrometer. Gas chromatography was used to determine the concentration of volatile organic compounds: EtOH, methanol, acetaldehyde, ethyl acetate, and higher alcohols. Samples were tested for 3 heavy metals-arsenic, cadmium, and lead. We used the guidelines developed by the Alcohol Measures for Public Health Research Alliance (AMPHORA) of the European Commission to assess risk. RESULTS: ABV ranged from 35.7 to 61.4%, and 58 of the 61 samples exceeded 40% ABV. The concentration of methanol, ethyl acetate, lead, arsenic, and cadmium was below AMPHORA guideline. The sum of higher alcohols exceeded the AMPHORA maximum in just 1 sample. Forty of the 61 samples had acetaldehyde levels beyond the AMPHORA guideline. CONCLUSIONS: The unrecorded Chinese alcohols we analyzed had a high EtOH concentration-a public health concern that is also presented by recorded alcohols. The high percentage of samples (65.5%) that had elevated acetaldehyde suggests the need to investigate the causes for this result and the need for steps to reduce acetaldehyde levels. The cumulative long-term risks of using high EtOH and high acetaldehyde Chinese spirits are heightened by the percentage of people in China who have a genetic trait for impaired acetaldehyde metabolism.

Stimulating the biosynthesis of antroquinonol by addition of effectors and soybean oil in submerged fermentation of Antrodia camphorata.

Antrodia camphorata is a precious medicinal mushroom that has attracted increasing attentions. Antroquinonol has been considered as being among the most biologically active components of A. camphorata. However, it was hardly biosynthesized via conventional submerged fermentation. Two approaches were applied to stimulate the biosynthesis of antroquinonol in submerged fermentation. On one hand, different kinds of effectors that may involve in the antroquinonol biosynthesis were investigated. Among the tested effectors, camphorwood leach liquor was the most effective for stimulating the antroquinonol production. On the other hand, because of the hydrophobic characteristics of antroquinonol, soybean oil was added to establish an extractive fermentation system for alleviating the product inhibition and resulting in enhanced productivity. The highest antroquinonol concentration could be achieved at 89.06 ± 0.14 mg/L when 10% (v/v) soybean oil was added at the beginning of the fermentation. This study will be of great significance for the study of A. camphorata and the bioprocess regulation of antroquinonol production.

Why solid-state fermentation is more advantageous over submerged fermentation for converting high concentration of glycerol into Monacolin K by Monascus purpureus 9901: A mechanistic study.

The underlying mechanisms by which solid-state fermentation (SSF) was more advantageous over submerged fermentation (SmF) for converting high concentration of glycerol into Monacolin K by Monascus purpureus were investigated innovatively. First, the established kinetic models and kinetic parameters showed that the cell growth, Monacolin K formation and glycerol consumption in SSF were more rapid than those in SmF. Secondly, the comparison of fatty acid composition of mycelial cells indicated a better fluidity and permeability of the cell membrane in SSF than that of SmF, which was also consistent with the difference in the ratio of extracellular/intracellular Monacolin K between the two systems. Thirdly, the phenomenon of glycerol concentration gradient was verified in SSF, which could well explain the resistance effect to high concentration of glycerol in SSF. These new findings provide some important insights to the elucidation of the advantages of SSF for the synthesis of fungal secondary metabolites.

Integrated strategy of pH-shift and glucose feeding for enhanced production of bioactive Antrodin C in submerged fermentation of Antrodia camphorata.

Antrodin C is one of the most potent bioactive components produced by the medicinal mushroom Antrodia camphorata. However, almost all studies in this field have focused on the biological activity of Antrodin C and relatively rare information has been reported regarding the biosynthetic process of Antrodin C. In this study, the strategies of pH-shift and glucose feeding for enhanced production of Antrodin C in submerged fermentation of A. camphorata were successfully applied in stirred bioreactors. The critical parameters for pH-shift and glucose feeding were systematically investigated. On one hand, the optimal culture pH for cell growth was distinct with Antrodin C biosynthesis and the maximum Antrodin C production was obtained by maintaining the first-stage culture at initial pH 4.5 and adjusted to 6.0 at day 8. On the other hand, it was beneficial for the Antrodin C accumulation with the initial glucose concentration of 40 g/L and feeding glucose to keep the residual sugar above 10 g/L. The maximum Antrodin C production (1,549.06 mg/L) was about 2.1-fold higher than that of control in 15-L stirred bioreactors by taking advantage of the integrated strategy of pH-shift and glucose feeding. These results would be helpful for the design of a highly efficient Antrodin C biosynthesis process.

Effect of cultural conditions on antrodin C production by basidiomycete Antrodia camphorata in solid-state fermentation.

Antrodia camphorata is a medicinal fungus and antrodin C is one of the main bioactive components of A. camphorata in the submerged fermentation (SmF). To optimize the culture conditions, the factors influencing the production of antrodin C by A. camphorata under solid-state fermentation (SSF) were investigated in this study. Different solid substrates and external nitrogen sources were tested for their efficiency in producing antrodin C. The response surface methodology was applied to evaluate the influence of several variables, namely, the concentrations of soybean meal, initial moisture content, and inoculum density on antrodin C production in solid-state fermentation. The experimental results show that the optimum fermentation medium for antrodin C production by A. camphorata was composed of 0.578 g soybean meal, 0.05 g Na2 HPO4 , 0.05 g MgSO4 for 100 g rice, with 51.83% initial moisture content, 22 day culture time, 28 °C culture temperature, and 35.54% inoculum density. At optimized conditions, 6,617.36 ± 92.71 mg kg(-1) yield of antrodin C was achieved. Solid-state fermentation is one good cultural method to improve the production of antrodin C by A. camphorata.

Enhanced production of Monacolin K by addition of precursors and surfactants in submerged fermentation of Monascus purpureus 9901.

The main problem in Monacolin K (MK) production by submerged fermentation of Monascus purpureus is low productivity. In this study, on one hand, addition of precursors was used to activate the biosynthesis of MK. When 4.0 g/L of sodium citrate was supplemented at the 48th H of the fermentation, the final MK production reached to 1,658.9 ± 28.5 mg/L after 20 day of fermentation, which was improved by 52.6% compared with that of the control. On the other hand, addition of surfactants could increase the permeability of cell membrane, thus driving more intracellular metabolites secreted into the fermentation broth and alleviating the product inhibition. When 40.0 g/L of Triton X-100 was added at the beginning of the fermentation, the final MK production reached to 2,026.0 ± 30.4 mg/L after 20 day of fermentation, which was improved by 84.9% compared with that of the control. These results are helpful to provide some new insights into the biosynthetic regulation on MK production; the approach can be applied to other fungal fermentation processes for enhancing production of useful metabolites.

Enhanced production of pigments by addition of surfactants in submerged fermentation of Monascus purpureus H1102.

BACKGROUND: The production of pigments by Monascus spp. has attracted increasing attention. Modification of the cell membrane structure by addition of surfactants has proved to be effective for the secretion of intracellular metabolites. Hence in this study the effects and underlying mechanism of surfactants on the production of pigments in submerged fermentation of Monascus purpureus H1102 were systematically investigated. RESULTS: Various surfactants exerted significant but different impacts on the biomass and production of pigments. The maximum production of pigment (304.3 U mL(-1) ) and highest extracellular/intracellular pigment ratio (1.46) were achieved when 15 g L(-1) Triton X-100 was added at 24 h of fermentation, corresponding to significant increases of 88.4 and 240% respectively compared with the control. Meanwhile, the concentration of citrinin (0.94 mg L(-1) ) was 20.6% lower than that of the control. A further study on the fatty acid composition of M. purpureus H1102 showed that the unsaturated/saturated fatty acid ratio and the index of unsaturated fatty acid increased significantly with the addition of Triton X-100. CONCLUSION: The addition of surfactant Triton X-100 could greatly enhance the production of pigment. It was suggested that Triton X-100 facilitated the secretion of intracellular pigment and therefore enhanced pigment production accordingly.

Efficient conversion of high concentration of glycerol to Monacolin K by solid-state fermentation of Monascus purpureus using bagasse as carrier.

High concentration of glycerol was used as the sole carbon source for efficient production of Monacolin K (MK) by solid-state fermentation (SSF) of Monascus purpureus 9901 using agricultural residue (bagasse), as an inert carrier. A comparative study showed that MK production in SSF was about 5.5 times higher than that of submerged fermentation when 26 % of glycerol was used, which may be due to the formation of glycerol concentration gradients in the inert carrier and less catabolite repression in SSF. For enhancement of MK yield in SSF, the effects of different influential variables, such as glycerol concentration, nitrogen source and its concentration, initial moisture content, inoculum size and particle size of bagasse, were systematically examined. All the factors mentioned above had an effect on the MK production in SSF to some extent. The maximal yield of MK (12.9 mg/g) was achieved with 26 % glycerol, 5 % soybean meal, 51 % initial moisture content, 20 % inoculum size and 1 mm particle size of bagasse. The results in this study may expand our understanding on the application of SSF using agricultural residue as carrier for production of useful microbial metabolites, especially the efficient conversion of high concentration of glycerol to MK by Monascus purpureus.

Changes in volatile compound composition of Antrodia camphorata during solid state fermentation.

BACKGROUND: Although the volatiles present in mushrooms and fungi have been investigated by many researchers, including Antrodia camphorata in submerged fermentation, there are few data available regarding changes in volatile compounds during fermentation. Our research has revealed that solid state fermentation of A. camphorata is highly odiferous compared with submerged cultures and the odor changed with increasing culture time. Therefore the aim of this study was to investigate the changes in volatile compound composition of A. camphorata during solid state fermentation. RESULTS: Altogether, 124 major volatile compounds were identified. The volatile compounds produced by A. camphorata during growth in solid state fermentation were quite different. Oct-1-en-3-ol, octan-3-one and methyl 2-phenylacetate were predominant in exponential growth phase production, while the dominant volatiles produced in stationary phase were octan-3-one and methyl 2-phenylacetate. In stationary phase, lactone compounds in A. camphorata, such as 5-butyloxolan-2-one, 5-heptyloxolan-2-one, 6-heptyloxan-2-one, contributed greatly to peach and fruit-like flavor. Terpene and terpene alcohol compounds, such as 1-terpineol, L-linalool, T-cadinol, (E, E)-farnesol, ß-elemene, cis-α-bisabolene and α-muurolene, made different contributions to herbal fresh aroma in A. camphorata. Nineteen volatile sesquiterpenes were detected from solid state fermentation of A. camphorata. The compounds 5-n-butyl-5H-furan-2-one, ß-ionone, (-)-caryophyllene oxide, aromadendrene oxide, diepi-α-cedrene epoxide, ß-elemene, α-selinene, α-muurolene, azulene, germacrene D, γ-cadinene and 2-methylpyrazine have not hitherto been reported in A. camphorata. CONCLUSION: The preliminary results suggest that the aroma-active compounds produced by A camphorata in solid state fermentation might serve as an important source of natural aroma compounds for the food and cosmetic industries or antibiotic activity compounds. The sesquiterpenes could be identified as possible taxonomic markers for A. camphorata.

Promotion of Monacolin K production by Agrobacterium tumefaciens-mediated transformation in Monascus albidus 9901.

The binary vector pCAMBIA3300-gpdA-hph-trpC with hygromycin B phosphotransferase (hph) was constructed and transformed into Monascus albidus 9901 by Agrobacterium tumefaciens-mediated transformation, with gene hph as the selective marker. In order to improve the efficiency of A. tumefaciens-mediated transformation in M. albidus 9901, we optimized various factors including concentration of M. albidus 9901 spores, cell density of A. tumefaciens, co-cultivation time, temperature, and acetosyringone concentration. Most transformants of M. albidus 9901 could grow stably on media containing 50 µg ml⁻¹ hygromycin B up to five generations. The presence of hph was identified by PCR. Two transformants H1 and H2 which produced more Monacolin K than M. albidus 9901 were screened, and the concentration of Monacolin K in the fermented millet by H1 and H2 increased by 42.15% and 40.34% respectively compared with that produced by M. albidus 9901.

[Use of ultrasensitive time-resolved fluoroimmunoassay for rapid detection of clenbuterol hydrochloride in urine].

OBJECTIVE: In order to provide a rapid and selectivity method for the determination of clenbuterol(CBL), an indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed. METHODS: Anti-CBL antibody, was raised by immunization against CBL-BSA in rabbits. CBL-OVA was coated by physical adsorption onto the microtitre plate, CBL or sample with CBL as a competitor. Both them were incubated with limited anti- CBL antibody. and a goat antirabbit IgG-Eu3+ conjugate was used as a tracer. RESULTS: The sensitivity of CBL-TRFIA was 0.01microg/L, and the recovery rate was 99.7%. RSD of CBL-TRFIA was 3.9% . The sensitivity of CBL-TRFIA provided a linear response from 0.01 - 25microg/L, with ED50 of (1.47+/-0.11) microg/L or ED80 of (0.07+/-0.01)microg/L and ED, of (23.6+/- 0.56) microg/L. The cross reactivity of the CBL-TRFIA with salbutamol, epinephrine hydrochloride and epinephrine bitartrate was negligible, while that with isoprenaline hydrochloride was 0.01% . Both CBL-TRFIA and CBL-ELISA test were applied for the quantitative measurement of CBL in the same urine, and the coefficient of correlation was 0.932. CONCLUSION: The CBL-TRFIA could be applied to detect the CBL in urine and it is useful to screening easily for CBL contamination in meat or foods.

[Natural occurrence of citrinin in Monascus products].

OBJECTIVE: In order to survey the natural occurrence of citrinin in Monascus products. METHODS: A total of 114 samples with either solid or liquid phases, collected from markets or delivered by the food production factories were analyzed by HPLC for citrinin. RESULTS: The results revealed that 68 (59.65%) samples were positive for citrinin with the levels between 0.18 and 1739.23 mg/kg (211.61 mg/kg for the average and 6.62 mg/kg for the median, respectively). The concentration of cirtrinin in various Monascus products is different. Twenty-five red pigment samples of Monascus were heavily contaminated with citrinin at the level ranged from 0.85 - 1739.93 mg/kg (average 508.40 mg/kg, median 169.88 mg/kg). Twelve of 19 red rice samples fermented by Monoscus species were not citrinin-free but 83.33% (10/12) with the citrinin levels below 10 mg/kg. The level of citrinin in 84% (21/25) health meals of Monascus is less than 6 mg/kg. CONCLUSION: The domestic Monascus products were contaminated by citrinin and the ratio of color value/citrinin in 15 (13.16%) samples exceeds the Japanese national standard.

[Study on the production of citrinin by Monascus strains used in food industry].

In order to screen strains with less or nearly no production of citrinin, thirty-five Monascus strains used in food industry were selected to investigate the effect of cultivation condition and the medium composition on citrinin production. The results from the study indicated that all strains produced citrinin on the rice with the levels ranging from 0.28 to 2458.80 mg/kg (201.60 mg/kg for the average and 61.99 mg/kg for the median, respectively), while 30 strains (85.71%) yielded this toxin on the submerged culture with the concentration between 0.09 and 55.65 mg/kg (11.99 mg/kg for the average and 3.51 mg/kg for the median, respectively). Therefore, citrinin production in rice in this study was higher than that in the liquid. In addition, the red pigment production in rice was 3-509 (average 93) times higher than that in the liquid. One strain with the highest color value (1134 U/g) but lower citrinin production in rice was obtained. These results suggested that it is necessary to make the safety evaluation of microorganisms for the production of foods and food ingredients, to investigate the ability of citrinin production by Monascus strains preserved by either the food manufacturer or the national culture collection units and, to survey the citrinin contamination in Monascus products countrywide. It is urgent for China to establish the tolerance limit of citrinin in foods fermented by Monascus species.