Impact Of Chinese Medicinal Herb On Solid-state Fermentation By Lovastatin-producing Monascus

Lovastatin is a key active substance in lipid-lowering functional red koji product. It can competitively inhibit activity of HMG-CoA reductase, a key enzyme during cholesterol synthesis, to effectively reduce serum lipids. Lovastatin in red koji is derived from secondary metabolism by Monascus spp.. Study on the fermentation strategies to regulate lovastatin production in red koji is a research hotspot in the field of functional red koji preparation. In recent years, Chinese medicinal herbs(CMHs) have been effectively used as additives to regulate fermentation from edible and medicinal fungi, which provides a new strategy to regulate red koji fermentation. Currently, CMHs used in the fermentation of red koji only involved enzymes, pigments, biomass and other indicators. However, no paper involved lovastatin was published, which greatly hampered the applications of CMHs in the regulation of lovastatin red koji fermentation.This study began with screening high-yield strains of lovastatin from different red koji samples. Then, investigated the influences of various edible-medicinal herbs on solid-state fermentation by Monascus spp.. At last, by quantitatively analysing expression patterns of lovastatin biosynthetic genes in response to different herb addition strategies, the impacts on Monascus spp. producing lovastatin influenced by CMHs was elucidated. The main achievements are as follows:1. One strain with the highest production of lovastatin named M2-1 was obtained from 11 red koji samples by lovastatin-resistant plate screening and solid-state fermentation verification. The yield of lovastatin reached 1.472 mg/g after 12 d fermentation. The strain was identified as Monascus ruber by morphological, biochemical and rDNA-ITS sequence analysis.2. Effects of powder and water extracts from 15 edible-medicinal herbs on M2-1 in solid-state fermentation were studied. According to the final results, Pericarpium Citri Reticulatae, Fructus Crataegi, Folium Mori, Radix Angelicae Dahuricae powder and Fructus Crataegi, Flos Caryophylli, Fructus Piperis water extract improved the quality of red koji significantly(P<0.05). By adding Pericarpium Citri Reticulatae powder and Fructus Crataegi water extract, the highest lovastatin production and color value of red koji reached at 2.118 mg/g and 320.12 U/g, which were increased by 43.89% and 71.79%, respectively. Set lovastatin production as experimental indicator, the optimal additive amount of each herb mentioned above were obtained by single factor experiment, and 3 herbs with significant effect were screened using Plackett-Burman experiment. Then, trial center was determined according to steepest ascent experiment. Finally, the optimum herb formula(3.75% Pericarpium Citri Reticulatae powder, 2.55% Fructus Crataegi powder and 2.01% Radix Angelicae Dahuricae powder) was acquired by Box-Benhnken experiment. Lovastatin yielded up to 3.601 mg/g by adding herbs according to the herb formula, which was increased by 1.45 times than the control without herb addition.3. Quantitatively analysed expression patterns of 6 lovastatin biosynthetic genes of M. ruber in response to different herb addition strategies during fermentation by q-PCR, and fermentation process parameters were also determined in the same process. The results showed that mixed herbs and Pericarpium Citri Reticulatae significantly up-regulated gene mokA, mokB, mokF, mokH and mokI on day 4, 6, 10 and 12, but they had no significant effect on mpleaA gene; Poria down-regulated the genes mentioned above in different degrees during the whole fermentation process. This results, combined with fermentation process parameters changed in response to different herb addition strategies, indicated that herbal consituents could regulate the expression level of lovastatin biosynthetic genes of M. ruber, then further regulated the phenotypic yield of lovastatin. The average level of relative gene expression showed that herbal consituents on the one hand indirectly regulated the transcription of mokA and mokB through preliminarily affecting the regulatory gene mokH, providing raw structural materials for lovastatin synthesis; on the other hand, herbal consituents could also directly regulate the mokF gene encoding transferase to synthesize lovastatin from structural materials. In addition, mokF gene may induced expression of mokI gene encoding efflux pump protein to secrete lovastatin from cystolic, which was helpful to remove feedback inhibition, thus maintained substrate utilization and metabolism level of M. ruber cells.