Solid-state Fermentation for Production of Monacolin K
on Soybean by Monascus ruber
Xiao-Qin Jia, Bao-Jun Xu, Mi-Young Lim, Chang-Keun Sung
(Department of Food Science and Technology, Chungnam Natl. University,
Daejon 305-764, Korea)
Abstract：Monacolin K was produced by solid-state fermentation on soybean using Monascus ruber GM011. Cultivation was carried out by incubation of mycelium liquid culture on soybean in 500-ml flasks. Effects of temperature and inoculum size of hyphae on monacolin K production were studied. The optimal temperature for monacolin K solid-state production was at 28°C, while the higher yield was obtained by cultivation at multiple-level-temperature than by that at single-level-temperature. The optimum inoculum size of mycelium culture was 15%, which reached to the high yield of total monacolin K, 4.094 mg/g dry soybean product after 20 days fermentation. Acid form monacolin K is the main in the fermented soybean all though fermentation stage, while lactone form monacolin K is below 25%. No citrinin can be detected by HPLC and competitive enzyme immunoassay.
Key words: Monascus ruber, solid-state fermentation, soybean, monacolin K
The filamentous fungal genus Monascus has been used to prepare red fermented product as a traditional medicine in China and other Asian countries for centuries. It can produce many bioactive secondary metabolites including Monascus pigments, monacolin K, Gama-Aminobutyric acid, unsaturated fatty acids and so on. Monascus fermented product might service as a functional dietary supplement for improving our blood circulation. The content of monacolin K is one of the criteria for value the fermented food.
Till now, almost all the functional Monascus fermented products were reported cultivated on rice medium. In this work, we tried this filamentous fungal fermentation on soybean to expand its application.
2 Materials and methods
2.1. Microorganism and fermentation
The monacolin K high producing fungus, GM011, was isolated from Chinese traditional food, red fermented rice. It was identified as Monascus ruber by its cultural characteristics and internal transcribed spacer (ITS) sequence comparison with type species in our previous work.
The stocked culture, M. ruber GM011, was maintained on Difco PDA (potato dextrose agar). After inoculation from the original slant, the dishes were incubated at 30°C for 6 days and subsequently stored at 4°C.
A two-stage fermentation technique was applied to produce Monascus secondary metabolites, which included seed culture stage (liquid culture in shake flask for mycelium growth) and metabolite production stage (solid-state fermentation on soybean for monacolin k production).
The fungal hyphae and spores were harvested by washing the petri dish cultures with a sterile aqueous solution of 0.1% Tween-80. The resulting suspension was used to inoculate the liquid seed medium, containing 40 g glucose, 40 ml glycerol, 10 g yeast extract, 1.5 g KH2PO4, 0.5 g MgSO4·7H2O in 1000 ml tap water. The seed culture was carried out at 30℃ for 18 h in replicated shake flasks held on a rotary platform shaker (150 rpm).
The metabolite production procedure was carried out in 500-ml Erlenmeyer flasks for 20 days. The preparation of soybean medium for solid-state fermentation was as follows: soybean, purchased from a local market, was soaked in tap water for 12 h. After drained off water, 60 g soaked soybean was autoclaved for 30 min at 121℃ and cooled to room temperature. Liquid seed was inoculated on the steamed medium.
2.2. Analytical methods
Commercial pure lovastatin (lactone form monacolin K) and citrinin were obtained from Sigma (St. Louis, MO., USA). All solvents used in chromatography were of HPLC grade; solvents used for spectroscopy were of spectroscopic grade, all other reagents used were of analytical grade. Trifluoroacetic (TFA) was purchased from Sigma (St. Louis, MO, USA). Water was of Milli-Q quality.
Monacolin K was determined as both its acid form and lactone form. Commercial pure lactone form monacolin K (LFMK) was used to prepare the acid form standards (AFMK) for the HPLC analyses. The standard solution of 500 μg/ml LFMK was converted to that of 500 μg/ml AFMK by adding l% 0.1N NaOH, heating at 50°C for 20 min. The resulted solution was held at 4°C until needed.
2.2.2. Sample extraction and clean up
The whole fermented soybean in the Erlenmeyer flask was collected and dried in 60 °C oven for 24 h. After ground into powder, 0.2 g fermented soybean was weighed and extracted with 1 ml extracting solvent, acetonitrile / water (60:40 by volume), by sonicating for 20 min at 20°C. The extraction was centrifuged at 12,000 g for 5 min at 4°C.After transferring the supernatant, extracting the residue with 1 ml extractingsolvent was repeated for other 4 times. These 5 times extraction solutions were combined and made up to 5 ml with the extracting solvent. After filtration (0.45 μm), the solutions were used for RT-HPLC analysis of monacolin K and citrinin.
2.2.3. RP-HPLC Analysis of Monacolin K and citrinin
HPLC was performed on a Hypurity C18 ODS (250 mm×4.6 mm i.d., 5 μm) column, which was mounted in a Beckman System Gold liquid chromatograph equipped with a Beckman diode array detector.
For monacolin K analysis, the mobile phase was a mixture of acetonitrile and 0.05% TFA (62:38, by volume) at a flow-rate of 0.8 ml/min. The UV detector was set at 237 nm.
For citrinin analysis, the mobile phase was a mixture of acetonitrile and 0.05% TFA (58:42, by volume) at a flow-rate of 0.5 ml/min. The UV detector was set at 334 nm.
2.2.4. Competitive enzyme immunoassay of citrinin
Ridascreen® Fast citrinin kit (R-Biopharm, Germany) was used for citrinin analysis. The microtiter plate spectrophotometer was Benchmark Microplate Reader (Bio-rad, U.S.A.) equipped with Bio-rad interference filter.
The competitive enzyme immunoassay (EIA) was carried out according to Ridascreen® Fast citrinin’s instructions. The absorbance values of reaction solution were measured at 450 nm.
3 Results and Discussion
3.1. Monascus fermented soybean and secondary metabolites
By the two-stage fermentation technique, Monascus ruber GM011 could be cultured on solid-state soybean medium (Fig. 1). After 20 days cultivation, the mycelium could grow all over the surface of soybean. It could be also observed that the inner of fermented soybean had turned red for the excretion of Monascus pigments by mature hyphae. Both AFMK and LFMK could be detected in the fermented soybean after 20 days cultivation in this research work; while, no citrinin could be detected either by RT-HPLC or by EIA methods, which mean lower detection limits were 2 µg/g (ppm) and 15 μg/kg (ppb) respectively.
Fig. 1 Monascus fermented soybean
Fig. 2 Typical chromatogram of Monascus fermented soybean for simultaneous analysis of both AFMK and LFMK. The retention times of AFMK and LFMK were 9.867 min and 15.233 min respectively.
3.2. Effect of temperature on monacolin K productivity
Liquid seed (10 ml mycelium culture) was incubated with steamed soybean at different temperatures for solid-state fermentation. Experiments at single-level-temperature (continuous temperature all through the fermentation stage) showed that the optimal temperature for monacolin K production is 28 °C; higher temperature (30 °C and 32 °C) can promote mycelium growth and decrease monacolin K yield, no LFMK could be detected at 30 °C and 32 °C cultivation, which is possibly for too low yield. Higher yield was reached by multiple-level-temperature procedure, which means incubation at 32°C for the first 3 days and then incubation at 28°C for the rest. The following fermentations were all by this method.
■: total monacolin K; △: AFMK; ○: LFMK
3.3. Effect of inoculum size on monacolin K productivity
Different sizes of inocula (10%, 15%, 20%, 25%) were administered to the steamed soybean (60 g). The result showed that small size (10%) means slow growth rate and secondary metabolite rate, while too big size (20%, 25%) mean low yield of secondary metabolites. Size of 15% showed the highest yield of total monacolin K, 4.094 mg/g after 20 days fermentation.
■: total monacolin K; △: AFMK; ○: LFMK
3.4. Effect of initial pH on monacolin K productivity
Monascus ruber can grow on acidic substrate, which is used to inhibit some bacteria contamination. Acidic substrate (HCl solution soaked) can cause to higher MK yield at first two week fermentation than nature substrate (pH 7.0 water soaked), while the later seemed better in a longer course. LFMK yield is always higher in acid substrate than that in nature one.
■: total monacolin K; △: AFMK; ○: LFMK
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