Fermentation of brown seaweeds by Monascus spp. for pigments and lovastatin production and characterization of biofunctional properties with fermented products

Abstract

Seaweeds are renewable plentiful marine biomass. In Korea, China, Japan, seaweed have been cultured and taken as traditional food. Both freshwater and marine algae produce many bioactive compounds with potential health benefits. The first study approache was aimed to maximize the production of natural pigments from the fermentation of Saccharina japonica as a solid state substrate by Monascus spp. at optimized physical and nutritional conditions. The yield of yellow, orange, and red pigments of Monascus purpureus were 83.01, 80.07, and 79.87 AU gdfs-1 and Monascus kaoliang were 83.23, 77.22, and 75.09 AU gdfs-1, respectively providing external 0.8 % glucose and 0.4 % peptone at incubation temperature 30 °C for 20 days. Medium inoculum size of 3 mL and moisture content of 50 % for M. purpureus and 60 % for M. kaoliang showed the highest pigments production. Scanning electron microscopic view showed better mycelial growth of M. purpureus than M. kaoliang on S. japonica. Monascus spp. fermented seaweed was highly efficient showing DNA protection and antioxidant activity because of increasing phenolic and flavonoid contents during fermentation period. The highest reducing sugar content was found at 15 days and glucanase enzyme activity was found at 20 days of fermentation for both of the species. The pigments were separated by thin layer chromatography and the Rf values were obtained 0.91 and 0.84 for yellow and orange pigments and two spots of 0.69 and 0.41 for red pigments. At different physico-chemical stress, red pigment of M. purpureus showed more stability than that of M. kaoliang. So, it can be concluded that S. japonica was a very suitable substrate at optimum conditions for Monascus spp. natural pigments production.

In the third chapter, two species of brown seaweeds, Saccharina japonica and Undaria pinnatifida were fermented by the red molds; Monascus purpureus and Monascus kaoliang to increase their bio-functional properties. The phenolic contents of S. japonica fermented by M. purpureus (SjMp) and M. kaoliang (SjMk) were the highest 71.53 ± 2.25 and 66.50 ± 4.64 mg gallic acid equivalent/g, respectively, whereas the highest flavonoid content was evident in S. japonica fermented by M. purpureus (SjMp) and U. pinnatifida fermented by M. purpureus (UpMp) (27.93 ± 0.28 and 26.88 ± 1.24 mg quercetin equivalent/g, respectively). Reducing sugar, protein and essential fatty acids levels also increased in fermented seaweeds. The antioxidant activities of fermented seaweed extracts exhibited significantly (p < 0.05) lower IC50 values than those of unfermented extracts. S. japonica fermented by M. purpureus (SjMp) exhibited the lowest IC50 values of antidiabetic activities mediated by α-amylase and rat intestinal α-glucosidase (maltose and sucrose): 0.98 ± 0.10, 0.02 ± 0.07 and 0.08 ± 0.13 mg/mL, respectively. M. purpureus fermented S. japonica extract at 4.58 ± 0.85 μg/mL afforded 50% inhibition of lipase, which was the most effective of all samples tested in this regard. Extracts from brown seaweeds fermented by Monascus spp. exhibited increased phenolics and flavonoids contents associated with strong in vitro bio-potential, DNA protection and the absence of any toxic effect on intestinal epithelial Caco-2 cells. Thus, fermented seaweed extracts may be recommended as food ingredients or therapeutic diet for patient suffering from oxidative stress, hyperglycemia and hyperlipidemia.

In the fourth chapter, Saccharina japonica (brown seaweed) was fermented in solid state by Monascus purpureus to maximize lovastatin production, the response surface methodology (RSM) was applied to optimize four different fermentation parameters: temperature (25−35 ºC), time (10−20 days), glucose (0.1−1.5%) and peptone (0.1−0.7%) concentration. The predicted combination of process parameters yielding the highest rate of lovastatin production (13.40 mg gdfs−1) was obtained at a temperature of 25.64 °C, a fermentation time of 14.49 days, glucose concentration of 1.32% and peptone concentration of 0.20%, with 92.85% validity. Among the studied factors, glucose, incubation time and temperature most strongly influenced lovastatin production. Triplicate experiments in which lovastatin was produced under optimal conditions resulted in a mean yield of 13.98 mg gdfs−1 with 207.84 mg gdfs−1 biomass. Liquid chromatography–tandem mass spectrometry (quadrupole time-of-flight) analysis confirmed the ionic molecular weight of lovastatin (405.26). Lovastatin from the M. purpureus fermented S. japonica exhibited thermal stability, superoxide dismutase activity and a cholesterol esterase inhibition activity that was higher than that of unfermented sample and showed no toxic effect on Caco-2 cell. Thus, S. japonica was a suitable substrate to maximize lovastatin production from M. purpureus at optimum condition for applying in food and pharmaceutical industry. The study showed that the Monascus-fermented Saccharina japonica containing lovastatin sample effect on 3T3L-1 cells to prevent preadipocyte to adipogenic differentiation. 3T3L-1 cells were treated with 500, 250 and 100 µg/mL of samples to prevent adipogenesis. The Oil red O staining and triglyceride measurement showed that adipocyte differentiation reduced in dose dependent manner. The mRNA gene expression was measured by Q-PCR to measure the expression level of adipogenic gene. The real time PCR result showed that the C/EBPα and PPARγ expression is reduced in lovastatin containing sample than unfermented seaweed. The gene expression of adipogenic gene such as LPL, Leptin, aP2, FAS and SRBP1 also reduced than control. Furthermore, the cell cycle of the 3T3L-1 cells also arrested in G1/S phase which is expressed by the reduced gene expression of Cyclin A, Cyclin D1 and CDK4. At last, the results showed that Monascus-fermented brown seaweed effectively control the adipogenic gene expression and inhibit lipid accumulation. So, the seaweed based Monascus-fermented food containing lovastatin could be a promising source of biofunctional food in food and pharmaceuticals industry.

This fifth chapter evaluated the immunomodulatory effects of Monascus-fermented Saccharina japonica extract on anti- and pro-inflammatory cytokines gene expression of THP-1 cells were evaluated. Extracts of fermented samples showed higher phenolic, flavonoid, protein and reducing sugar contents than unfermented one. Fermented samples were rich in many bioactive compounds determined by GC-MS analyses and showed cell viability greater than 85% in MTS assay. Regarding the anti-inflammatory and pro-inflammatory activities of the different samples, Q-PCR analyses revealed that IL-10 gene expression in THP-1 cells was significantly higher (p < 0.05) in cells treated with the SjMp or SjMk sample than those treated with the unfermented sample. Cells treated with the SjMp extract or lipopolysaccharide (LPS) showed significantly (p < 0.05) higher relative gene expression of IL-4 cytokine than cells treated with SjMk or SjU extracts. The relative gene expression of IFN-α was higher in cells treated with SjMp followed by LPS, SjMk and SjU. TGF-β expression was higher in LPS-stimulated cells followed by SjMk and other samples. Cells treated with SjMp exhibited significantly higher pro-inflammatory (IL-6, IL-8, TNF-α and NF-kB) cytokine gene expression than cells treated with SjU. These results revealed that extracts from S. japonica fermented with Monascus spp. regulate cytokine gene expression.

The sixth chapter was conducted to evaluate the antioxidant and kinetics of α-amylase, α-glucosidase inhibition of ethanol extract from Monascus spp. fermented Saccharina japonica containing pigments. Marine biomass, Saccharina japonica was utilized as a solid-fermented substrate for Monascus spp. fermented ethanolic extract production. The ethanolic extract of S. japonica fermented by M. purpureus (SjMp) showed DPPH, ABTS+, SOD and DNA protection activities of 92.82%, 99.40%, 79.33% and 89.47%, respectively at 10 mg/mL concentration. The pigments rich ethanolic extracts of fermented sample showed significantly higher (p<0.05) antioxidants, pancreatic α-amylase and intestinal α-glucosidase inhibition activities than unfermented one. IC50 values were significantly (p<0.05) lower in ethanolic extract of fermented sample than unfermented one. Kinetic analysis revealed that ethanolic extract of fermented sample inhibited the α-amylase competitively but α-glucosidase displayed both competitive and non-competitive inhibition. The Linewaver-Burk kinetics analysis revealed that ethanolic extract of S. japonica fermented by M. kaoliang (SjMk) showed higher Km value (4.55 mg) than ethanolic extract of SjMP (2.74 mg) followed by ethanolic extract of SjU (0.35 mg) at 10 mg/mL concentrations as α-amylase inhibitor. But incase of α-glucosidase inhibition, the Km value was higher in ethanolic extract of SjMp (2.30 mg) with Vmax (0.29 mg/min) at 10 mg/mL concentrations. The toxicity analysis of fermented and unfermented samples showed no toxic effect on Caco-2 cells. The amino acid compositional analysis by Thin Layer Chromatography (TLC) showed that fermentation process cause change in free amino acids contents. Pigment rich Monascus-fermented ethanolic extract showed positive antibacterial activity on Aeromonas hydrophila and Staphylococcus aureus. So, Monascus spp. fermented S. japonica extract played an important role in prevention of free radicals formation and hyperglycemia which can be applied in biofunctional food formulation.