Introduction

A fungal species, Monascus, has been used for traditional fermented food and has attracted attention because of its biologically active metabolite production, such as monacolin K (lovastatin), and γ-amino butyric acid (GABA) [1]. Monacolin K is a secondary metabolite of Monascus strains, and Endo [2] discovered that M. ruber produces an active methylated form of compactin known as monacolin K (lovastatin; mevinolin) in liquid fermentation. Monacolin K functions as an inhibitor of 3-hydroxy-3-methyl glutaryl-coenzyme A reductase, which is a regulatory and rate-limiting enzyme of cholesterol biosynthesis [2]. GABA has several physiological functions, such as neurotransmitting, hypotensive, and diuretic effects, which were reported to be affected by culture conditions either in submerged or solid cultures [3].

Monascus-derived fermented foods have been also characterized by its deep-red color; therefore, they are called red-yeast rice or red-mold rice. Monascus strains have long been used for the production of pigments that have been typically used, particularly in East Asia, as a coloring agent for foodstuffs [4].

In contrast to the advantage of Monascus as a food microorganism, some strains of Monascus were reported to produce citrinin, a nephrotoxin, which was previously found in Aspergillus and Penicillium genera and might contaminate in Monascus-derived food. Repression of citrinin production in Monascus has been reported using genetic disruption [5], culture condition optimization, and mutation [6].

Based on the background of Monascus sp. as a functional food fermentant, fungi expressing Monacolin and/or GABA with a deep-red color, but no citrinin, would be desirable; however, the correlation between the pigment and citrinin production is not clear.

In this study, we screened for a suitable Monascus fungus for fermented food with high lovastatin and low citrinin production with a deep-red color, followed by an examination of red koji using two-step temperature-shift cultivation.

Materials and Methods

Fungal Strains and Cultivation

Twenty-nine strains of Monascus spp. were used in this study, as listed in Table 1. To maintain the culture, strains were grown on a potato dextrose agar (PDA, Difco, CA, USA) slant followed by storage at 4 °C. For koji (rice mold) preparation, ca. 50 g (for Table 2), or 1 kg (Fig. 2), of moistened rice in 200-ml Erlenmeyer flasks were steamed with autoclave at 121 °C for 5 min, followed by the inoculation of 1 ml conidial suspension (5 × 105-conidia/ml) of Monascus spp. were inoculated to the rice with 4 ml of sterilized deionized water. They were then incubated at 30 °C for 4 days, followed by further incubation at 25 °C for additional 17 days (Table 2) or 10 days (Fig. 2), with daily mixing aseptically. YPD medium (Difco, CA, USA) was prepared for genomic DNA preparation. Unless otherwise stated, cultivations were carried out at 30 °C.

Table 1 Monascus strains used in this study.
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Table 2 Summary of citrinin, lovastatin, and pigment production by Monascus spp.
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Genetic Manipulations

Genomic DNA from Monascus spp. were extracted from YPD medium-grown mycelium with ISOPLANT II (Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. The PKS homologue gene was amplified by polymerase chain reaction (PCR) with a primer set designed from the CTpks gene of M. purpureus (Genbank accession no. AB243687), CTpksf GACACGGCATGCTCGTCAT and CTpksr ACTCACCAAAGCTGTGCCC under the following conditions: 95 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min, and a final elongation step at 72 °C for 10 min.

Sequencing

PKS homolog genes amplified by PCR were subcloned in pT7-blue vector (Takara-bio, Shiga, Japan) followed by sequencing on both strands by the vector primer with the ABI model 3100 and Big Dye terminator kit (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer’s instructions.

Analysis

Cellular growth at various temperatures was examined by measuring the colony diameter grown on PDA plate. For citrinin and pigment analysis, 20–50 g of the koji was sampled, followed by the crushing with Oster Blender ST-1 (OSAKA CHEMICAL Co.,Ltd., Osaka, Japan) for 2 min, extraction with 2.5 volume of 70% ethanol with extensive voltexing for 1 h. Total pigment production was determined as described by Tseng et al. [7]. Citrinin was analyzed using a RIDA screen FAST citrinin kit (Adumax Co., Tokyo, Japan) based on ELISA. Lovastatin was measured by HPLC using a Shimadzu 10A (Shimadzu Co., Kyoto, Japan) under the following conditions: column: Wakosil-II 5C18 with 4.6 mm ID and 250 mm length (Wako Pure Chemicals Co., Tokyo, Japan); oven temperature, 40 °C; flow rate, 0.5 ml/min, after isocratic elution with methanol—0.1% phosphate (72:28, v/v) for 25 min followed by linear gradient from methanol—0.1% phosphate (72:28, v/v) to methanol—0.1% phosphate (100:0, v/v) until 30 min; UV detection at 238 nm. All the data were determined by at least duplicate analysis.

Results and Discussion

Selection of Fungi Producing High Lovastatin and High Pigments but No Citrinin

In order to select Monascus spp. suitable for added-value fermentation seeds, we selected using three indexes, lovastatin, pigment, and citrinin productions. Figure 1 shows the profiles of the examined strains with regard to pigmentation and citrinin production in solid culture. There was no clear correlation between the pigment (absorbance at 500 nm) and citrin production. Table 2 summarizes the results of the selection using citrinin, pigment, and lovastatin production for the strains used in this study. Of the 31 strains tested, 16 strains exhibited citrinin production less than 1,000 μg/g koji under the conditions in this study and subjected to lovastatin production. Ten of the 16 produced lovastatin and four strains produced higher than 100 μg/g koji.

Fig. 1
figure 1

Relationships between citrinin production and pigment production among various Monascus spp. Error bars represent the standard deviation from the mean value of duplicate experiments

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The gene responsible for citrinin production, PKScit, was further amplified to identify the correlation between citrinin phenotypic production and the detected genes. PKScit was amplified from genomic DNA of M. purpureus and M. kaoliang with identity higher than 97%. Since there has been only one report on PKScit from M. purpureus [5], other genes could have high similarity in M. kaoliang while low similarity in M. ruber and M. pilosus. It is also reported that M. ruber and M. pilosus are evolutionarily closer than M. purpureus by phylogenetic analysis using β-tublin sequences [8].

Based on the results described above, as strain M. pilosus NBRC4520 showed the highest lovastatin production with comparable pigment production, we selected strain M. pilosus NBRC4520 for further study.

Production of Lovastatin by Temperature Shift Solid Fermentation

Separation of cellular growth and production phase could contribute to significantly increase productivity. Preliminary growth test showed that the strain NBRC4520 grew from 20 to 42 °C, and the optimal temperature was found to be 30 °C (data not shown). After solid culture was carried out at 30 °C for 4 days, each culture was transferred to various conditions, as shown in Table 3. Although anaerobic conditions produced no lovastatin, cultivation at 23 °C under aerobic conditions increased production after 6 days, compared to at 30 °C, the optimal temperature for cellular growth. It was previously reported that lovastatin, citrinin, and GABA productions were affected by various culture conditions including temperature. In M. purpureus NTU601, the highest lovastatin production was obtained at 30 °C, while no examination with temperature-shift cultivation was carried out [9]. It is also noted that lower temperature (25 °C) gave less lovastatin productivity in the strain NTU601, while higher production at 23 °C than that at 30 °C in case of our study.

Table 3 Lovastatin produced by strain NBRC 4520 at various temperatures.
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Figure 2 shows the detailed time course of temperature-shift cultivation under aerobic conditions in koji (rice mold) preparation in a large scale. After shifting the temperature from 30 to 23 °C on the 4th day, lovastatin started to be produced linearly until the 12th day with pigment production. On the 14th day, lovastatin production reached 225 μg/g dry koji without producing citrinin. No citrinin production in this case may be due to the shorter cultivation time than that for Table 2 or caused by scaling up from 50 g to 1 kg. This should be further examined using the 1 kg production scale. In contrast, no lovastatin was detected also in this scale when the temperature was constant at 30 °C, as expected from data in Table 3 (data not shown). Regarding the effect of physical culture conditions for Mosascus spp., the effect of light irradiation was reported [10]. Their study shows that red light enhanced GABA, red pigments, monacolin K, and citrinin, in a submerged culture of M. pilosus and M. purpureus. In a solid culture, nutrients enhancing secondary metabolites of Monascus spp. have been extensively investigated, such as sodium nitrate [3], water and ethanol [10]. In the large scale production, lovastatin production nearly stopped at 225 μg/g dry koji while higher productivity was obtained in a small scale (565 μg/g dry koji in Table 2). Examinations for further increased production are currently in progress either by nutrients feeding or operational conditions optimization.

Fig. 2
figure 2

Time course of citrinin, lovastatin, and pigment production by M. pilosus NBRC 4520 during koji (rice mold) cultivation. Circles: citrinin (μg/g dry koji), triangles: pigment (Abs at 500 nm), and squares: lovastatin (μg/g dry koji). Temperature was changed from 30 to 23 °C at 4 days (arrow). Error bars represent the standard deviation from the mean value of triplicate experiments

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Conclusion

Temperature-shift cultivation has been applied as a simple technology to improve production in submerged culture [11]. Here, we report the possibility of temperature-shift cultivation for lovastatin production by Monascus spp. Initial cultivation at optimal growth temperature followed by preferential lovastatin production at lower temperature, suppressing cellular growth, could also be applied for other Monascus spp. exhibiting higher lovastatin productivity. We are currently examining the details of condition optimization, such as nutrient supply, to improve the production of both lovastatin and pigment.