Production of red mold rice using a modified Nagata type koji maker

In this research, a commercial koji maker with a rotary perforated bed of 5-m diameter was modified for red mold rice production. Monascus purpureus BCRC 31499 was selected for its high production capacities of monacolin K and red pigment. The selected strain was first cultivated in a 120-l submerged type fermentor at 34°C and 2 vvm aeration rate with 60 rpm agitation for 5 days using 20% liquefied rice porridge as carbon source. The high concentration red mold rice broth (>3.5 g/ml) was harvested for inocula and well mixed with cooked rice to an initial concentration of 2% v/w. The inoculated cooked rice then was directed into the modified koji maker, in which temperature and humidity profiles were kept at varied levels at different stages, respectively. Air was circulated to remove fermentation heat while the perforated bed rotated slowly for providing mild agitation. Lag phase of the Monascus sp. in the modified koji maker was determined to be 16 h by the time the koji temperature raised rapidly. Water was added into the koji bed by a water curtain at the 36th hour to keep the moisture content of the rice koji at 50% or above. At the final stage, temperature was adjusted to 34°C to direct red pigment production. After 7 days, 1,200-kg high quality red mold rice was harvested per batch. Labor costs, space, and fermentation time were reduced tremendously compared with those made by traditional methods.     

Effect of red mold rice on antifatigue and exercise-related changes in lipid peroxidation in endurance exercise

This study evaluated the effect of red mold rice supplementation on antifatigue and exercise-related changes in lipid peroxidation of male adult Wistar rats through swimming exercise. Thirty 16-week-old rats were studied by dividing them into three groups (ten for each group). Other than the control group (CD), the other two groups were divided into a high-dose (HD) treatment group (5 g red mold rice/kg body weight for the HD group), and a low-dose (LD) group (1 g red mold rice/kg body weight for the LD group). Swimming endurance tests were conducted after 28 days of red mold rice supplementation, and the result showed that the treatment group showed a higher exercise time (CD, 78.0±6.4; LD, 104.2±9.6; and HD, 129.4±10.9 min; p<0.05) and a higher blood glucose concentration (CD, 76.67±8.08; LD, 111.34±8.50; and HD, 117.67±11.06 mg/dl; p<0.05) than the CD. Moreover, the blood lactate (CD, 45.00±0.90; LD, 31.41±1.80; and HD, 28.89±1.62 mg/dl; p<0.05), blood urea nitrogen (CD, 21.87±0.75; LD, 20.33±0.83; and HD, 20.53±1.09 mg/dl; p<0.05), and hemoglobin (CD, 14.20±0.21; LD, 13.70±0.55; and HD, 13.28±0.35 g/dl; p<0.05) were also significantly lower than those of the CD. Besides, the result suggested that the red mold rice supplementation may decrease the contribution of exercise-induced oxidative stress and improve the physiological condition of the rats.     

Association mapping of grain color,phenolic content,flavonoid content and antioxidant capacity in dehulled rice

Phytochemicals such as phenolics and flavonoids in rice grain are antioxidants that are associated with reduced risk of developing chronic diseases including cardiovascular disease, type-2 diabetes and some cancers. Understanding the genetic basis of these traits is necessary for the improvement of nutritional quality by breeding. Association mapping based on linkage disequilibrium has emerged as a powerful strategy for identifying genes or quantitative trait loci (QTL) underlying complex traits in plants. In this study, genome-wide association mapping using models controlling both population structure (Q) and relative kinship (K) were performed to identify the marker loci/QTLs underlying the naturally occurring variations of grain color and nutritional quality traits in 416 rice germplasm accessions including red and black rice. A total of 41 marker loci were identified for all the traits, and it was confirmed that Ra (i.e., Prp-b for purple pericarp) and Rc (brown pericarp and seed coat) genes were main-effect loci for rice grain color and nutritional quality traits. RM228, RM339, fgr (fragrance gene) and RM316 were important markers associated with most of the traits. Association mapping for the traits of the 361 white or non-pigmented rice accessions (i.e., excluding the red and black rice) revealed a total of 11 markers for four color parameters, and one marker (RM346) for phenolic content. Among them, Wx gene locus was identified for the color parameters of lightness (L*), redness (a*) and hue angle (H ^o). Our study suggested that the markers identified in this study can feasibly be used to improve nutritional quality or health benefit properties of rice by marker-assisted selection if the co-segregations of the marker–trait associations are validated in segregating populations.     

Red mold fermented products and Alzheimer's disease: a review

Alzheimer's disease is seen mainly in individuals over the age of 65, and the morbidity rate increases with age. Regarding the health function of Monascus-fermented red mold rice (RMR), besides hypolipidemic and hypotensive effects, other health functions of RMR such as anti-oxidation, cancer prevention, anti-fatigue, and anti-obesity have also been reported. Many published studies have shown the efficacy of RMR in the prevention of Alzheimer's disease. The current article discusses and provides evidence to support the beneficial potential of RMR in the prevention of Alzheimer's disease by discussing the pathogenic factors of Alzheimer's disease and the secondary metabolites of Monascus.     

In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia

Monascus or more commonly known as red mold rice is fermented rice on which Monascus purpureus has been grown. It has been a traditional Chinese food additive for thousands of years in China. Secondary metabolite product of Monascus, monacolin K, has been proven that it could be used as an antihypercholesterolemic agent. In this study, M. purpureus NTU568 mutated and selected from a monacolin K productivity strain—M. purpureus HM105 produced high quantities of monacolin K at a level of 9,500 mg kg⁻¹. This research focused on the effect of adding red mold rice powder of M. purpureus NTU568 to a hamster diet on total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-C). In the results, the oral administration of Monascus powder in hyperlipidemia hamster was indeed proven to decrease TC, TG, and LDL-C levels. Plasma TC levels in hamster fed with Monascus powder at onefold dosage [10.78 mg (day 100 g bw)⁻¹] for 4 and 8 weeks were significantly lower (31.2 and 22.0%, respectively) than that in hyperlipidemia hamster. Plasma TG (30.1 and 17.9%) and LDL-C levels (36.0 and 20.7%) were also significantly lowered by feeding Monascus powder at onefold dosage for 4 and 8 weeks compared to hyperlipidemia hamster. In addition, examinations of liver TC and TG levels of hyperlipidemia hamster were also performed and showed similar effects on lipid-lowering action by oral administration of Monascus powder. Since citrinin is a mycotoxin that possesses nephrotoxic and hepatoxic effects, it has a negative impact on the safety of red mold rice for people. This study examined the liver somatic index [plasma glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) levels] and liver biopsy to investigate whether Monascus powder induced damage in liver. It was found that the plasma GOT and GPT levels were not significantly increased by feeding Monascus powder. There was no difference in the results of the liver biopsy between the Monascus powder-treated groups and the control group.     

Spread of herbicide‐resistant weedy rice (red rice,Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide‐resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F₂ or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.     

Antioxidative Pyranonigrins in Rice Mold Starters and Their Suppressive Effect on the Expression of Blood Adhesion Molecules

Antioxidants having a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity in rice mold starters, which are used for the preparation of various Japanese fermented foods, and their effectiveness against the expression of blood adhesion molecules were examined. An antioxidant was isolated from the rice mold starters used for shochu and identified as pyranonigrin-S (PG-S) by ¹H-NMR, ¹³C-NMR, and FAB-MS analyses. It was a derivative of pyranonigrin-A (PG-A), which has been isolated as an antioxidant from the rice mold starters. Pyranonigrins PG-A and PG-S were found to exist in spores on rice mold starters which had been prepared by Aspergillus awamori, A. kawachii, and A. saitoi. PG-S exhibited a higher level of DPPH radical scavenging activity than PG-A. PG-A was found to have a significant suppressive effect on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) (P<0.05).     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Monascus secondary metabolites: production and biological activity

The genus Monascus, comprising nine species, can reproduce either vegetatively with filaments and conidia or sexually by the formation of ascospores. The most well-known species of genus Monascus, namely, M. purpureus, M. ruber and M. pilosus, are often used for rice fermentation to produce red yeast rice, a special product used either for food coloring or as a food supplement with positive effects on human health. The colored appearance (red, orange or yellow) of Monascus-fermented substrates is produced by a mixture of oligoketide pigments that are synthesized by a combination of polyketide and fatty acid synthases. The major pigments consist of pairs of yellow (ankaflavin and monascin), orange (rubropunctatin and monascorubrin) and red (rubropunctamine and monascorubramine) compounds; however, more than 20 other colored products have recently been isolated from fermented rice or culture media. In addition to pigments, a group of monacolin substances and the mycotoxin citrinin can be produced by Monascus. Various non-specific biological activities (antimicrobial, antitumor, immunomodulative and others) of these pigmented compounds are, at least partly, ascribed to their reaction with amino group-containing compounds, i.e. amino acids, proteins or nucleic acids. Monacolins, in the form of β-hydroxy acids, inhibit hydroxymethylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis in animals and humans.     

Application of Cyanidin-3-Glucosides as a functional food ingredient in rice-based bakery products

BackgroundBlack pericarp rice has recently become popular among rice consumers for its diverse health benefits specially anti-cancer effect. Cyanidin-3-Glucosides (C3G), an prominant bioactive component of anthocyanins which is abundantly present in black pericarp rice.ObjectivesWe investigated, how effectively it can be used to fortify Cyanidin-3-Glucosides (C3G) content in red and white pericarp polished rice or rice based bakery products for more nutritional value.MethodIn the present study, we have characterized several black pericarp rice cultivars along with some red pericarp and white pericarp rice cultivars by physicochemical including mineral profiling, and quantified the C3G by UFLC and LCMS.ResultsC3G content was significantly reduced from raw rice to cooked rice condition. All the black pericarp rice cultivars synthesized C3G, while this content was not detected in red and white pericarp rice cultivars. However, when 25% of black pericarp rice were mixed with 75% red or white pericarp polished rice, C3G content was significantly retained in cooked rice conditions. Formulation of rice-based bakery food product using black pericarp rice powder was also remarkably retained the C3G content as compared to that of cooking. Black rice is harder in texture, difficult to digest and needs higher energy for cooking. Therefore, we tried to circumvent these challenges by fortifying 25% of black pericarp rice with white or red pericarp rice.ConclusionFortification of C3G enriched black rice (25%) with red or white pericarp rice (75%) might bring a better nutritional quality in both cooking and baking condition. This may lead a way to the effective management of the non-communicable disease such as cancer for common rice consuming population.     

Statistical analysis and validation of process parameters influencing lovastatin production by Monascus purpureus MTCC 369 under solid-state fermentation

Monascus, a fermented rice (red mold rice), was found to reduce total cholesterol and triglyceride in serum due to the presence of lovastatin, a 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor. Optimization and validation of different process parameters such as temperature, fermentation time, inoculum volume, and pH of the solid medium was done using Box-Behnken’s factorial design of response surface method for maximum production of lovastatin by Monascus purpureus MTCC 369. A maximum lovastatin production of 3.422 mg/g was predicted by day 14.43 of fermentation in a rice based solid medium of pH 6 when fermented at a temperature of 29.46°C, an inoculum volume of 5.11 mL, and using response surface plots and the point prediction tool of Design Expert 7.1.3 (Statease Inc., USA) software.     

CRISPR/Cas9‐mediated functional recovery of the recessive rc allele to develop red rice

Red rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health‐promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14‐bp frame‐shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9‐mediated method to functionally restore the recessive rc allele through reverting the 14‐bp frame‐shift deletion to in‐frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T₁ in‐frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild‐type and in‐frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14‐bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame‐shift alleles for crop improvement.     

Silicon Alleviates Drought Stress of Rice Plants by Improving Plant Water Status,Photosynthesis and Mineral Nutrient Absorption

Drought is a major constraint for rice production in the rainfed lowlands in China. Silicon (Si) has been verified to play an important role in enhancing plant resistance to environmental stress. Two near-isogenic lines of rice (Oryza sativa L.), w-14 (drought susceptible) and w-20 (drought resistant), were selected to study the effects of exogenous Si application on the physiological traits and nutritional status of rice under drought stress. In wet conditions, Si supply had no effects on growth and physiological parameters of rice plants. Drought stress was found to reduce dry weight, root traits, water potential, photosynthetic parameters, basal quantum yield (F _v/F ₀), and maximum quantum efficiency of PSII photochemistry (F _v/F _m) in rice plants, while Si application significantly increased photosynthetic rate (Pr), transpiration rate (Tr), F _v/F ₀, and F _v/F _m of rice plants under drought stress. In addition, water stress increased K, Na, Ca, Mg, Fe content of rice plants, but Si treatment significantly reduced these nutrient level. These results suggested that silicon application was useful to increase drought resistance of rice through the enhancement of photochemical efficiency and adjustment of the mineral nutrient absorption in rice plants.     

OsRAN2, essential for mitosis,enhances cold tolerance in rice by promoting export of intranuclear tubulin and maintaining cell division under cold stress

With global climate change, abnormally low temperatures have affected the world's rice production. Many genes have been shown to be essential for molecular improvement of rice cold‐tolerance traits. However, less is known about the molecular cellular mechanism of their response to cold stress. Here, we investigated OsRAN2 involved in regulation of cell division during cold stress in rice. Expression of OsRAN2 was increased under cold treatment, but not during salt and drought stress. The mean root mitotic index was closely related to the expression level of OsRAN2. Knockdown transgenic rice lines showed an aberrant organization of spindles during mitosis and stunted growth during development. Overexpression of OsRAN2 enhanced cold tolerance in rice. The transgenic rice overexpressing OsRAN2 showed maintained cell division, decreased proportion of cells with intranuclear tubulin and formation of a normal nuclear envelope under the cold condition. Our study suggests a mechanism for OsRAN2 in regulating cold resistance in rice by maintaining cell division through promoting the normal export of intranuclear tubulin at the end of mitosis. This insight could help improve the cold‐tolerance trait in rice.     

Effects of Thai black sticky rice extract on oxidative stress and lipid metabolism gene expression in HepG2 cells

Anthocyanins, which are found in some food, including Thai black sticky rice, are reported to have health-promoting properties. Oxidative stress plays a major role in the pathogenesis of many degenerative diseases induced by free radicals, such as cardiovascular disease, stroke and cancer. We evaluated the anthocyanin-rich extract (ARE) from Thai black sticky rice for antioxidative and antihyperlipidemic effects on HepG2 cells. Cell viability was investigated with the neutral red assay and the MTT assay, and oxidative stress was determined by the DCFH-DA assay. RT-PCR was used to evaluate the effect of ARE on LDLR, HMG-CoAR, PPAR (α1,γ) and LXRa gene expression. We found that ARE at high doses (≥ 800 mg/L) induces cytotoxicity. However, at 600-1000 mg/L it reduced intracellular oxidative stress (P < 0.05) in a dose-dependent manner, and at 200 mg/L it significantly enhanced the expression of the LDLR gene in HepG2 cells. We concluded that ARE can be beneficial for health promotion by reducing oxidative stress and enhancing LDL clearance, regulating LDLR production on the cell surface membrane, thereby maintaining lipid homeostasis.     

Improvement of monacolin K, γ-aminobutyric acid and citrinin production ratio as a function of environmental conditions of<Emphasis Type="Italic"> Monascus purpureus</Emphasis> NTU 601

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and -aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30°C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.     

Hypersensitivity of SV40-Transforme Cells to the Action of Tunicamycin

The manufacturing processes used determined the physicochemical properties of the three kinds of rice food, garaeduk, bagsulgi, and cooked rice. The initial rate of hydrolysis by porcine pancreatic α-amylase (PPA) was affected by the food form. The firmer structure of garaeduk was apparently responsible for the difficulty in maceration, resulting in less digestion than with easily digestible food for the same maceration time. The initial rate of hydrolysis of each rice product by PPA increased with increasing maceration time in a Waring Blender for all of the processed rice products. The postprandial glucose and insulin responses to the three processed rice products were also studied in ten patients with type 2 diabetes mellitus (4 men and 6 women aged 56.8±2.3 yr; duration of diabetes, 3.6±1.2 yr; body mass index (BMI), 23.7±2.6 kg/m²; fasting serum glucose, 143.9±5.1 mg/dl; serum insulin, 20.8±2.2 μU/ml). Each subject ingested of the three rice foods after a 12-h overnight fast, and the serum glucose and insulin levels were measured over a 0–240 min period. The postprandial serum glucose and insulin levels at 90 min after ingesting bagsulgi and cooked rice were less than those at 60 min, while the levels at 90 min after ingesting garaeduk were higher than those at 60 min. Garaeduk also significantly decreased the incremental responses of glucose and insulin when compared with bagsulgi and cooked rice. The results suggest that garaeduk would be the most unlikely to increase the postprandial serum glucose and insulin levels among the three rice foods. The food form, which eventually differentiated each food by its specific surface area with the same degree of maceration because of the characteristic physical strength, therefore affected the rate of rice starch hydrolysis both in vitro and in vivo.