Monascus is a small filamentous saprophytic fungus and can be grown by inoculation into rice for fermentation, converting the rice to ‘red yeast rice’. Red yeast rice is a traditional Chinese medicine that has been used in Asian food and therapeutic applications for nearly a thousand years. Its applications include coloring, preservation of traditional foods, and treatment of indigestion, diarrhea and spleen-stomach disharmony. Monascus fermentation can produce functional substances such as pigments, monacolin K (MK) and γ-aminobutyric acid , but most Monascus strains can also produce citrinin that exhibits liver and kidney toxicity . The presence of citrinin limits the food application of Monascus to a certain extent.
MK, also known as lovastatin, was first obtained by separation from M. ruber fermentation products in 1979 by the Japanese professor Akira Endo . MK inhibits cholesterol synthesis by competitively inhibiting the activity of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), thus lowering cholesterol levels in human and animal blood. The study found at least 14 substances with hypolipidemic function in red yeast rice, including MK, monacolin J, monacolin L, monacolin M, monacolin X and their acidic structures, dehydroMK, dihydromonacolin L and compactin .
Common fermentation modes for Monascus to produce MK include solid state fermentation (SSF) and submerged fermentation (SmF). In fermentation optimization studies for MK production by Monascus, most research has focused on SSF methods because they offer has many advantages compared to SmF methods. Fungi can produce more enzymes and secondary metabolites in SSF, and some enzymes can only be produced in SSF . Zhang et al. [6, 7] utilized SSF by adding agar to a liquid medium, then inoculating Monascus into the solid medium cut into small pieces, and used a liquid medium without agar for fermentation comparison. Compared with Monascus SmF, biomass accumulation, MK synthesis rate, glycerol consumption rate, cell membrane fluidity and permeability, and glycerol tolerance concentration in Monascus under SSF conditions were significantly higher. Furthermore, MK yield of Monascus under SSF is usually several to dozens of times higher than that under SmF [8, 9].
Scholars have previously reviewed the research progress in lovastatin production by Aspergillus terreus fermentation . Herein, we comprehensively review the research progress in MK production using Monascus fermentation processes, from the breeding of high-yield MK strains to the optimization of fermentation medium and fermentation conditions, including SSF and SmF methods.
2 Monascus strain breeding for high-yield MK production
Monascus strain choice has an important influence on production of MK by fermentation, and the ability of different strains to produce MK under the same culture conditions varies greatly. Usually, mutagenesis of Monascus is performed using separation and screening [11, 12, 13] or by physical or chemical methods to obtain strains with high yield of MK, thereby significantly increasing MK yield and reducing citrinin content. Suh et al.  obtained multiple mutants with high MK yields through γ-ray treatment, among which the KU609 mutant had the highest MK yield, nearly 10 times higher than the original strain. After optimization of culture conditions, MK yield of KU609 can reach 977 mg/kg. There are also studies on mutagenesis of Monascus strains by combining ultraviolet (UV) irradiation and LiCl treatment. UV irradiation for 45s was performed under 1.0% LiCl conditions, then five mutant strains were obtained after three rounds of irradiation. M. purpureus ZT35, a strain with high MK yield, was obtained through five generations of culture, which has a MK yield that is three times higher than that of the original strain . In addition, MK synthesis by Monascus has a competitive relationship with the synthesis of red pigment. Studies have shown that treatment of Monascus spores with UV irradiation or ethyl methanesulfonate (EMS) can result in an albino strain that does not produce red pigment and citrinin, and its MK yield is significantly higher than that of the original strain .
3 Optimization of fermentation medium for MK production by Monascus
Due to the two fermentation modes of Monascus, SSF and SmF, and the significant difference in the composition of medium between SSF and SMF , this paper reviews the optimization of SSF and SMF medium components respectively.
3.1 Optimization of solid medium components
The types of substrates and nutrient additives were optimized to increase the MK production by Monascus SSF (see Table 1 for details).
Optimization of the SSF Medium Components of Monascus.
|strain||nutrients||optimal nutrients||MK production (mg/kg)||references|
|Monascus Mutant KU609||Nitrogen source: Histidine, Yeastextract, Glutamic, Valine Tyrosine, Alanin, Lysine Peptone, Soytone Carbon source: Glucose, Fructose, Maltose, Lactose, Glycerol, Sucrose, Galactose Inorganic salts: MgSO4, MnSO4, FeSO4, ZnSO4, NaNO3, CaSO4, CuSO4||2.5% Soytone, 2.4% Glucose and 0.26% MgSO4||977.70||Suh et al. |
|M. purpureus KCCM 60168||Nutrients: Glucose, Peptone||1.32% glucose and 0.20% peptone||13,400||Suraiya et al. |
|M. pilosus KMU108||Nutrients: Ganghwayakssuk, Glucose, Sucrose, Lactose, Galactose||2.2% ganghwayakssuk and 3.8% glucose||3,007||Lee and Lee |
|M. purpureus TISTR 3541||Nutrients: Sodium nitrate, Methionine, Glycerol||2% glycerol, 0.14% methionine and 0.01% sodium nitrate||5,900||Jirasatid |
|M. purpureus 9901||Nitrogen sources: peptone, yeast extract, Soybean meal, Ammonium sulfate, Sodium nitrate Carbon source: glycerol||5% soybean meal and 26% glycerol||12,900||Lu et al. |
|M. purpureus CMU002UXX-32-44||Nutrients: NH4Cl, MgSO4·7H2O||4 mg/g NH4Cl and 0.2 mg/g MgSO4·7H2O||6,238.2||Kanpiengjai et al. |
|M. pilosus MS-1||Nutrients: Soybean flour, Water content, Acetic acid, MgSO4·7H2O||35% water content, 0.6% (v/w) acetic acid and 0.004 mol/kg MgSO4·7H2O||18,733||Feng et al. |
|M. sanguineus||Nutrients: Soybean, CaCl2, Acetic acid||20 g/L soybean, 2.5 g/L CaCl2 and 25 μL acetic acid||20,040||Dikshit and Tallapragada |
|M. purpureus MTCC 369||Inorganic salts: NH4Cl, MgSO4.7H2O, NaCl, CaCl2.2H2O||14.32 g/L NH4Cl, 0.76 g/L MgSO4, 14.65 g/L NaCl and 0.54 g/L CaCl2||3,403||Kraboun et al. |
|M. ruber||Carbon source: Glycerol Nitrogen sources: Beef extract, Peptone, Soybean powder, Yeast extract, Ammonium sulfate, Sodium nitrate||20% glycerol and 3% soybean meal||19,8100||Zhang et al. |
3.1.1 Solid matrix
The type of solid matrix is an important factor affecting fungal growth and metabolite production. Rice is a commonly used matrix for Monascus fermentation [25, 26]. However, to develop a matrix more suitable for MK production than rice, researchers have used various solid matrixes for Monascus fermentation and compared their MK yield (see Table 2 for details). Zhang et al.  carried out solid-state fermentation using M. ruber with different kinds of grains such as rice, millet, wheat and barley. The results showed that MK yield was the highest in millet matrix (7.25 mg/g), probably due to the low viscosity and small particles of millet, while wheat (2.45 mg/g) and barley (2.69mg/g) had poor water absorption, therefore hyphae were not easy to grow, resulting in a lower MK yield.
Comparison of MK Yield by Monascus Fermentation Using Different Matrixes.
|Strain||Matrix||Optimal Substrate||MK Production (mg/g)||References|
|M. ruber||Rice, millet, corn, barley, wheat||Millet||7.25||Zhang et al. |
|M. purpureus CMU002U||Purple rice (Doi Muser; Doi Saked; Na; Nan; PHayao; Hom CMU)||Doi Muser||13.48||Pengnoi et al. |
|M. purpureus NTU 301||Dioscorea, sweet potato, cassava, rice, potato||Dioscorea||2.58||Lee et al. |
|M. purpureus MTCC- 410||Raw rice, parboiled rice, wheat, barley, finger millet, germinated finger millet, maize, sorghum, njavara||Raw rice||0.64||Venkateswaran V. and Vijayalakshmi G. |
|M. purpureus MTCC 369||Besan flour, jaggery, palm jaggery, black gram, green gram, barley, sago, ground nut cake, sesame waste, millet, ragi, wheat bran, rice bran, jack fruit seed, jong grain rice,||barley||193.70||Seraman et al. |
|M. purpureus MTCC 410||Wheat bran, tamarind seed, jack fruit seed, rice bran||Wheat bran||0.094||Dikshit. et al. |
Different rice varieties also have significant effects on MK yield from Monascus fermentation. Pengnoi et al.  used different varieties of purple rice as the matrix for Monascus fermentation. The results showed that MK yield (13.48 mg/kg) of purple rice variety Doi Muser as a matrix was significantly higher than that of other varieties of purple rice.
The viscosity of rice has a great influence on MK yield. Because of its relatively high viscosity and easy agglomeration, glutinous rice is not conducive to Monascus growth . However, some researchers have studied Monascus fermentation with glutinous rice as a matrix, and obtained a considerable MK yield. Water content is the key control factor for fermentation with glutinous rice as matrix. Yu et al.  used rice and glutinous rice as substrates, and performed orthogonal design optimization of the fermentation conditions (initial water content, matrix size and fermentation time) of the two substrates. The results showed that initial water content had a large impact on MK yield fermented with the two substrates, and optimal MK yields of rice and glutinous rice were 2.50 mg/g and 2.71 mg/g, respectively. Meanwhile, water content is also closely related to fermentation time. During the fermentation process, water content will gradually decrease with water evaporation and utilization by microorganisms. Kanpiengjai et al.  fermented glutinous rice with an X-ray induced mutant strain M. purpureus CMU002UXX-32-44, and optimized its fermentation conditions. The results showed that MK yield could reach up to 6428 mg/kg under the conditions of 72.5% initial water content and 38 d fermentation time. Therefore, by effectively controlling water content of glutinous rice, it can also be considered an excellent matrix for MK production by fermentation using Monascus.
Other studies used non-cereal materials as the matrix for fermentation by Monascus. For example, yam can also be used as a matrix for fermentation. Yam has previously been associated with blood-lipid lowering effects, and red yeast yam after fermentation by Monascus has more significant blood fat-lowering effects than red yeast alone . Lee et al.  used cassava, sweet potato, potato, yam and rice as matrixes for fermentation, and found that MK content of M. purpureus strain NTU 301 grown on yam matrix can reach 2584 mg/kg, which is 5.37 times higher than MK yield from rice matrix (481 mg/kg). Suraiya et al.  and Hong et al.  used kelp and red ginseng, respectively, as matrixes for Monascus fermentation, and obtained high yields (13.98 mg/g, and 3.09 mg/kg, respectively)
3.1.2 Nutritional additives
Addition of carbon source (e.g. glucose, glycerol and lactose), nitrogen source (e.g. soybean meal, peptone and glutamic acid), inorganic salts and other additives to the solid matrix are effective methods to increase Monascus MK yields. Glucose can promote Monascus growth, which promotes the production of MK. Suraiya et al.  optimized the response surface methodology and found that the optimal glucose addition for Monascus solid medium was 1.32%. Suh et al.  also found during medium optimization experiments in mutant strain KU609, that adding 0.4 g of glucose to 20 g of rice could significantly increase its MK yield. Lee et al.  mixed 2.2% wormwood with 3.8% glucose and used it as a matrix for Monascus fermentation, finding that its MK yield was 6 times higher than that using rice as matrix.
Glycerol is also often used as a carbon source for MK fermentation of Monascus [6, 19]. Lu et al.  used bagasse as an inert carrier, and added different concentrations of glycerol (18%-34%) as a single carbon source to solid and liquid media for M. purpureus strain 9901. Researchers found that the optimal glycerol concentration (up to 26%) for MK production under SSF was significantly higher than under SmF, while MK yield under SmF decreased with increasing glycerol concentration and was significantly lower than that under SSF. The reason may be that cell morphology and physiological characteristics of Monascus under SSF are different from those under SmF, and the lower metabolic repression under SSF is more conducive to Monascus growth, thus the utilization rate of glycerol by thallus is also increased. Studies by Feng et al.  showed that when glycerol concentration was greater than 10%, metabolic repression of Monascus cells under SmF increased, and MK secretion was inhibited by varying degrees by increasing glycerol concentration.
Some studies have found that adding ethanol as a carbon source to medium can significantly increase MK yield and reduce citrinin content. Experimental results showed that the MK yield of Monascus was significantly higher than from a control group after adding 0.5% of ethanol by volume. In this study, a response surface method was used to optimize ethanol addition, culture temperature and water content. The optimal combination was: 500 g rice, 0.3% ethanol, culture temperature 30°C, 120 mL water. The MK yield under these conditions was 2.6 times higher than that of the control group, and citrinin content was reduced by nearly half . However, higher concentrations of ethanol affect Monascus polyketide synthase (PKS) activity, resulting in polyketide synthesis inhibition (including MK, red pigment and citrinin). When the ethanol concentration reached 4%, the yield of polyketides of Monascus was significantly reduced. On the other hand, the biomass of Monascus increased at 2% and 4% ethanol concentrations relative to the control group .
Nitrogen is an essential nutrient for the growth of fungal hyphae. At lower concentrations, NaNO3 is superior to organic nitrogen sources in increasing MK yield , but too high NaNO3 concentration is toxic to cells, resulting in a decrease in MK yield. Soybean meal is usually added to the culture medium as a nitrogen source. Xu et al.  mixed rice and soybean meal as a substrate. When the additive amount of soybean meal is 20%, MK yield can reach 4.02 mg/g, which is approximately 2 times higher than MK yield (1.26 mg/g) without soybean meal. Another study was conducted to optimize the response surface of medium for MK production by strain M. pilosus MS-1 SSF. It was found that when substrate was 60% rice and 40% soybean meal, MK yielded up to 17.66 mg/g . Dikshit et al.  also studied the additive amount of soybean meal by using wheat bran as a substrate in fermentation of strain M. sanguineus. The results showed that addition of 20 g/L soybean meal could significantly increase MK yield, and the highest yield was up to 20.04 mg/g. In addition, the addition of amino acids such as glutamate  and methionine  act as a nitrogen source, or precursor for MK in the medium and can increase MK yield. Kraboun et al.  studied the effects of varying sodium glutamate and peptone concentrations on MK yield from the Monascus fermentation process. Researchers found that sodium glutamate was superior to peptone in increasing MK yield under the same nitrogen content.
In many filamentous fungi, specific monovalent and divalent cations have important effects on promoting cell growth and production of metabolites. Panda et al.  used the strain M. purpureus MTCC 369 for SSF and optimized the inorganic salt components of the medium as a single factor. It was found that NH4Cl, MgSO4 and NaCl could greatly increase MK yield. The effects of MnSO4 and CaCl2 were secondary, while the effects of KH2PO4 and FeSO4 were poor. Subsequently, the four inorganic salts (NH4Cl, MgSO4, NaCl, CaCl2) were optimized using response surface methodology, and the obtained optimal combination was: NH4Cl 14.32 g/L, MgSO4 0.76 g/L, NaCl 14.65 g/L and CaCl2 0.54 g/ L. In addition, inorganic salts also have different effects on MK yields from different strains. Dikshit et al.  used M. purpureus and M. sanguineus strains to optimize SSF medium by Plackett-Burman design. MgSO4·7H2O significantly increased MK yields of the two strains, and MnSO4·7H2O also had a significantly promoted MK yield from M. sanguineus. In addition, MK yield from the two strains significantly decrease after the addition of CaCl2.
3.2 Optimization of liquid medium components
The medium components were optimized to increase the MK yield of Monascus SmF, such as carbon source, nitrogen source, inorganic salts, inducers and precursors (see Table 3 for details).
Optimization of the SmF Medium Components of Monascus.
|strain||nutrients||optimal nutrients||MK production (mg/L)||references|
|M. pilosus MK-1 M. pilosus||Carbon sources: Glucose, Maltose, Fructose, Glycerol, Lactose Nitrogen sources: Peptone||M. pilosus MK-1: 3% glucose, 7% glycerol and 3.8% peptone M. pilosus: 1% Maltose, 7% glycerol and 3.8% peptone||725 (M. pilosus MK-1) 444 (M. pilosus)||Miyake et al. |
|M. ruber CCRC 31535||Rice powder, Peptone, Glycerin, Glucose||34.4 g/L rice powder, 10.8 g/L peptone, 26.4 ml/L glycerin and 129.2 g/L glucose||131||Chang et al. |
|M. purpureus 410||Carbon sources: Glucose, Maltose, Fructose, Lactose, Sucrose Nitrogen sources: Peptone, Yeast extract, Ammonium chloride, Ammonium sulphate, Ammonium nitrate||4.4% maltose and 0.14% peptone||81.27||Mohan-Kumari et al. |
|M. purpureus MTCC 369||Dextrose, NH4Cl, KH2PO4, MgSO4·7H2O, MnSO4·H2O||29.59 g/L dextrose, 3.86 g/L NH4Cl, 1.73 g/L KH2PO4, 0.86 g/L MgSO4·7H2O and 0.19 g/L MnSO4||351||Sayyad et al. |
|M. purpureus MTCC 369||Glucose, Peptone, NH4Cl, KH2PO4, Yeast extract, K2HPO4, KNO3, MgSO4·7H2O, MnSO4·H2O, NaCl, CaCl2·2H2O, FeSO4·7H2O||52.61 g/L glucose, 16.65 g/L peptone, 1 g/L NH4Cl, 1 g/L KH2PO4, 3 g/L yeast extract, 1 g/L K2HPO4, 0.5 g/L KNO3, 0.2 g/L MgSO4·7H2O, 0.418 g/L MnSO4·H2O, 0.5 g/L NaCl, 0.1 g/L CaCl2·2H2O and 0.001 g/L FeSO4·7H2O||88.54||Seraman et al. |
|M. purpureus 9901||Precursors: Ethanol, Sodium acetate, Sodium citrate, Trisodium citrate, Methionine, Phenylalanine Surfactants: PEG-400, PEG-4000, Tween-20, Tween-80, Span-40, Span-80 and Trition X-100||4.0 g/L sodium citrate (supplemented at 48 h) and 40.0 g/L Triton X-100||2,026.0||Zhang et al. |
3.2.1 Carbon source
In SmF of Monascus, medium composition is crucial, and different nutrients have different effects on MK production in the thallus. Regarding the influence of carbon sources, there are inhibitory carbon sources (e.g. glucose), moderately inhibitory carbon sources (e.g. fructose and maltose) and non-inhibitory carbon sources (e.g. glycerol) . Glucose plays an important role in fermentation, and it can be rapidly absorbed and utilized by the thallus in a short time, thereby promoting mycelial growth of filamentous fungi. However, when glucose is used as a single carbon source, higher concentrations of glucose (>70g/L) will inhibit the expression of MK-related genes, resulting in a decrease in MK yield . Nevertheless, by combining glucose with different carbon sources, the inhibition of MK synthesis by glucose can be alleviated, thereby significantly increasing MK yield. Chang et al. [44, 50] combined higher doses of glucose (129.2 g/L) with appropriate levels (26.4 ml/L and 36.4 ml/L) of glycerol as a carbon source for Monascus fermentation, finding that MK yield was up to 131mg/L. Miyake et al.  used different carbon source combinations for a M. pilosus SmF process. The results showed that MK yield from fermentation with combined carbon sources was significantly greater than for a single carbon source. MK yield was significantly higher (444mg/L) when using a combination of maltose and glycerol 1:7 (v/v), than other combinations. A high glucose and glycerol condition (glucose/glycerol = 7:7) or high glycerol ratio (glucose/glycerol = 1:7) were not effective for Monascus production of MK, yielding 3.74 mg/L and 8.62 mg/L, respectively. An optimized glucose/glycerol ratio (3:7) could significantly increase MK production (108.43 mg/L) however. This highlights that the combination ratio of glucose and glycerol as a carbon source can significantly reduce the inhibitory effect of glucose on MK synthesis.
3.2.2 Nitrogen source
Nitrogen source is another important limiting factor that affects MK yield by regulating Monascus growth. Peptone is usually a suitable nitrogen source for MK production by Monascus SmF processes . Mohan-Kumari et al.  compared the effects of different nitrogen sources on MK yield from fermentation by Monascus, and found that MK yield where peptone was used as a nitrogen source was significantly greater than that of other nitrogen sources. However, MK yield is usually related to nitrogen source limitation in the stable phase. When microorganism growth is limited by the consumption of nitrogen source, redundant carbon sources are used for the production of secondary metabolites, thereby improving yield of secondary metabolism . Therefore, it is necessary to limit the nitrogen source. Miyake et al.  combined carbon source and nitrogen source in different proportions for fermentation by M. pilosus. The results showed that under different carbon source combinations, even though glucose that has a strong inhibitory effect on MK yield, the optimal carbon-to-nitrogen (C/N) ratio is between 7-9.
3.2.3 Inorganic salts
Inorganic salts are also essential in SmF by Monascus, such as metal ions such as Fe2+, Ca2+, Zn2+, Cu2+, Mg2+ and Mn2+, which have been shown to promote cell growth and MK synthesis of Monascus in SmF . Studies have shown that Mg2+ and Mn2+ have the most significant positive effect on MK production by Monascus SmF compared with other inorganic salts , and these two inorganic salts can even orchestrate their positive effects on MK synthesis when both are present . In addition, Plackett-Burman design was used in other studies to analyze the effects of different inorganic salts on MK yield from Monascus. The results showed that Mn2+ had the most significant effect on yield .
3.2.4 Inducers and precursors
The addition of linoleic acid also significantly increased MK yield from Monascus in SmF. As a quorum sensing molecule, linoleic acid can stimulate the cAMP-PKA pathway, thereby increasing the transcription level of MK synthesis-related genes, thus increasing MK yield. The addition of 512 μmol/L and 256 μmol/L linoleic acid significantly up-regulated the expression of mok A and mok H genes. When 512 μmol/L linoleic acid was added, the yield of MK was 1.35 times greater than that of the control group .
Studies have shown that the addition of precursors and surfactants can also increase MK yield from Monascus SmF. In the process of secondary metabolite biosynthesis, the precursor substance directly participates as a substrate in the synthesis of MK. Surfactant can alter the permeability of cell membranes and promote nutrient absorption and discharge of intracellular metabolites, thereby increasing MK yield . Zhang et al.  compared the effects of different precursors (ethanol, sodium acetate, sodium citrate, trisodium citrate, methionine and phenylalanine) on MK yield, and found that various precursors had different extents of promoting effects on mycelial growth of M. purpureus 9901 and its MK yield. Sodium citrate had the most significant positive effect on MK yield, and the optimal promoting effect was achieved when the concentration of sodium citrate was 4 g/L and addition time was 48 h after fermentation. In this study, the effects of different surfactants on cell growth of strain M. purpureus 9901 and MK synthesis were also investigated. The results showed that Triton X-100 could simultaneously increase biomass and MK yield from hyphae, and the effect was optimal when the concentration was 40 g/L.
4 Fermentation process optimization for MK production by Monascus
Figure 1 shows that the main parameters affecting the MK production of Monascus fermentation products are as follows: initial moisture content of the solid matrix, initial pH of the medium, inoculum size, culture temperature and fermentation time. The optimization of these parameters was one of the research hotspots for scholars to increase the MK production of Monascus (Table 4).
Comparison of MK Yield by Monascus Fermentation Under Different parameters.
|strain||fermentation parameters||optimal parameters||MK production (mg/kg)||references|
|M. purpureus MTCC 369 and M. ruber MTCC 1880 (co-culture)||Temperature, Fermentation time, Inoculum volume, pH of the solid medium||30°C, 14 d, 10% inoculum volume, pH=6||2,830||Panda et al. |
|M. sp. KB9||Temperature, Initial moisture, Initial pH||30°C, initial moisture content 38% (w/w), initial pH=7||13,536||Subsaendee et al. |
|M. purpureus||Initial Moisture content, Particle size, Fermentation time||initial moisture content 50%, particle size 4 cm, 13 d||2,500||Yu et al. |
|M. purpureus MTCC 369||Temperature, Fermentation time, Inoculum volume, pH of the solid medium||29.46°C, 14.43 d, inoculum volume 5.11 mL, pH=6||3,422||Panda et al. |
4.1 Culture temperature
Regardless of SmF or SSF, culture temperature is critical for the growth of filamentous fungi and the production of secondary metabolites. Usually Monascus can grow at 18-45°C, and its optimum culture temperature is 30-38°C . Studies have shown that the strain M. purpureus MTCC369 and M. ruber MTCC 1880 can obtain the highest MK yield under the constant temperature culture conditions of 29.46°C . However, some reports have shown different results. For example, Suraiya et al.  showed that the strain M. purpureus KCCM 60168 had the highest MK yield at a constant temperature of 25.64°C.
In microbial cells, the optimal temperature for different enzymes varies. With ongoing deepening studies on temperature, researchers have found that variable temperature culture has a significant effect on MK yield from Monascus. Tsukahara et al.  separated the growth stage of Monascus from the production stage of MK. The screened strain M. pilosus NBRC4520 with high-yielding MK, was first cultured at 30°C for 4 days, and then placed at 30°C or 25°C for culture. The results showed that MK productivity was low during solid state constant temperature incubation at 30°C, but MK yield was nearly 20 times higher than that of constant temperature culture after the culture temperature was reduced from 30°C to 25°C. Pengnoi et al.  also found that variable temperature culture contributed to MK production by Monascus in SSF. Under the tested temperature combinations, the yield of MK cultured at 30°C and then cultured at 25°C was the highest, which was 2.8 times and 1.4 times of that cultured at constant temperature of 30°C and 25°C respectively. Studies have found that constant temperature culture at higher temperature (30°C) is more conducive to the growth of strain M. fuliginosus CG-6 mycelium, and dry weight of cells under constant temperature culture is significantly higher than under variable temperature culture. At lower temperatures (25°C), MK synthetic gene cluster-related genes had higher expression levels than that at higher temperatures. Under variable temperature conditions, the expression level of efflux pump protein encoded by the mok I gene showed the same upward trend as the mok I transcription level in variable temperature culture. Hence, it is more favorable for Monascus to secrete MK at 25°C .
Mohan-Kumari et al.  also increased MK yield of Monascus SmF by using a variable-temperature culture. Monascus was first incubated at 30°C, and then transferred to different temperatures for culture. Results indicated that in variable temperature culture, Monascus cultured at 25°C in the late stage had the highest MK content in the fermentation broth, but the MK content in Monascus cells was lower, which affected total yield of MK. When cells were cultured at 28°C in the later stage, there was greater MK concentration in both in intracellular and extracellular domains, which increased total MK yield most significantly, and MK yield was increased by nearly 60% compared with a constant temperature culture.
4.2 Unitial water content of the solid matrix
In SSF, water content of the matrix is also an important factor. Water plays an important role in the nutrient utilization and physiological activities of Monascus. Substrates with lower water content are not conducive to Monascus growth, so MK yield is adversely affected in these conditions . Water content between 60%-70% can be optimal for the MK yield and biomass of the filamentous fungus. This may be due to the presence of sufficient oxygen and water in the substrate molecular layers, which supports fungal growth and metabolic heat removal. However, when the water content is increased from 80% to 90%, the oxygen in the matrix is replaced by water, leading to insufficient oxygen supply, so the biomass and MK yields from the thallus are reduced .
It noteworthy that the matrix particle size is an important factor affecting matrix water content. Generally, smaller substrate particles will provide a larger surface area for the attachment of microorganisms, which supports mycelial growth and metabolite accumulation . However, too small a substrate size often leads to substrate aggregation, thereby causing a decrease in inter-particle voids and an increase in oxygen transfer resistance, which is disadvantageous for mycelial growth and MK production [6, 61].
In addition, different strains have different tolerances to water content. When using rice as a substrate, the optimum water content of the strain Monascus sp. KB9 is 38% , and the optimal water content of the strain M. purpureus is 50% , while the optimum water content of strain Monascus sp. M12-69 is 55-75% .
4.3 Initial pH of the medium
In Monascus SSF, research shows differing results for optimum pH for MK production. Lee et al.  prepared yam as a solid substrate of varying pH (3, 7 and 9) for Monascus fermentation, and found that strain M. purpureus NTU 301 produced more MK under acidic conditions (pH=3), and MK yield decreased as the pH increased. However, it has also been reported that when rice is fermented by Monascus sp. KB9, MK yield is highest when the substrate pH is between 5-7 . Panda et al.  found that when rice was fermented by strain M. purpureus MTCC 369, the optimal pH of the matrix for MK production was 6. Therefore, the choice of optimal pH for the substrate may vary depending on the interaction between different strains, different substrates or other fermentation conditions.
The pH value of liquid medium also plays an important role in MK production by Monascus SmF. pH has a significant effect on the catalytic process of many enzymes and the transport of various components of the cell membrane, thus affecting the use of substrates by fungi. As reported in the literature, A. terreus has accelerated metabolism at higher pH (7.5) and accelerated the absorption and utilization of carbon sources, which increases MK yield . Mohan-Kumari et al.  found that acidity of liquid medium had a significant effect on the biomass and MK yield of strain M. purpureus MTCC 410, and the yield of MK under low acid fermentation conditions (pH=5) was significantly higher than that at other pHs. The reason is that too-high or too-low pH affects the production of key enzymes required for MK production, and under specific pHs, some enzymes that degrade MK may be activated, resulting in decreased final MK yield. In addition, comparison between biomass and MK yields at different pHs reveals that higher biomass also affects MK production. Therefore, MK yield can only be increased only by reducing excessive accumulation of Monascus biomass.
4.4 Inoculum size
The inoculum size of Monascus also has an effect on its MK yield, and the degree of influence varies for different strains. Studies have found that when the inoculum size of Monascus seed solution is 10-40% of the SSF medium, there is no significant difference in MK yield by fermentation of strain M. purpureus 9901 , while MK yield of strain M. ruber is significantly affected. When the inoculum size exceeds 20%, MK yields of the two strains decrease to varying degrees. The reason is that Monascus biomass accumulates rapidly under the big inoculum size, and MK yield decreases due to premature consumption of nutrients . Therefore, it’s better to use an inoculum size of Monascus up to 20%.
4.5 Fermentation time
The substrate concentration and fermentation conditions will affect the peak time of secondary metabolites. The MK yield of Monascus stabilizes when it is fermented to 12-18d [13, 24, 40], and the optimum fermentation time is usually 14-15d [17, 54, 56]. As the fermentation time goes on, MK content will reach the peak value followed by a decreasing trend [15, 62]. Therefore, it is recommended to control the fermentation time of MK production by Monascus to about 15d.
5 Conclusion and outlook
MK is a secondary metabolite produced by filamentous fungus Monascus, which can significantly lower cholesterol levels in the human body. The fermentation modes of Monascus include SSF and SmF. Generally, MK yield under SSF is several to ten times higher than that under SmF. For an increase of MK yield from fermentation by Monascus, methods such as strain selection and optimization of fermentation conditions are generally used. The most commonly used method for strain selection is to adopt a physical method such as UV irradiation or chemical method such as a mutagenic agent (e.g. LiCl) to induce mutagenesis, thereby obtaining a Monascus strain with high MK yield.
For SmF medium of Monascus, the choice and addition of carbon and nitrogen source is a key factor in MK yield. The combination of different carbon sources for fermentation helps reduce the inhibition effect from single carbon sources, such as glucose on MK synthesis, thereby increasing MK yield. MK yield is usually associated with nitrogen source limitation during the stable phase. When Monascus growth is inhibited by nitrogen source limitation, redundant carbon sources can be used for secondary metabolite production, thereby improving secondary metabolite yield. Therefore, it is necessary to effectively control C/N ratio of the medium, and the optimal ratio is 7-9. In addition, appropriate exogenous inducing substances (metal ions, linoleic acid, etc.), precursors (sodium citrate, sodium acetate, etc.) or surfactants can also increase MK yield of Monascus to some extent.
For SSF medium of Monascus, in addition to containing sufficient nutrients, the matrix should meet conditions such as low viscosity, low agglomeration, easy moisture absorption, appropriate water content and have small particles. In addition, nutrients such as glycerin and glucose as a carbon source, soybean meal and peptone as a nitrogen source, and metal ions such as Mg2+ and Zn2+ can significantly increase MK yield from Monascus.
In terms of fermentation conditions of Monascus, variable temperature cultures are useful for an increase of MK yield. The reason is that the growth and development of Monascus cells differs from MK synthesis-related enzymes by their optimum temperature, and growth and development and MK synthesis are subject to subsection adjustment by variable temperature cultures. First, Monascus are incubated at 30°C for 3-5 d, and then adjusted to approximately 25°C, which significantly increases MK yield. For initial pH of the medium, it is better to be controlled at 4~6. For optimum conditions the inoculum size of Monascus seed solution should not exceed 20% of the SSF medium, fermentation time should be maintained for approximately 15d, and initial water content of the solid matrix in SSF should be controlled at 50~70%.
Nowadays, the research and industrial production technology of MK production by Monascus SSF is mature, but the research and industrial application of SmF is relatively scarce. Compared with SSF, Monascus’ SmF has the advantages of lower labor intensity, better automation technology, stable product quality, short production cycle and low cost. Furthermore, SmF is more conducive to large-scale factory production and market competitiveness of fermentation products. Therefore, use of SmF for MK production will be an inevitable trend in the future production of MK by Monascus fermentation processes.
This study was supported financially by the International Science and Technology Cooperation and Exchange Project of Fujian Agriculture and Forestry University (KXGH17001), the Special Fund for Science and Technology Innovation of Fujian Agriculture and Forestry University (CXZX2018067 and CXZX2018068) and the Foreign Cooperation Projects of Fujian Science and Technology Program (2019I0008) in China.
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