Abstract

Monascus purpureus, a pigment-producing ascomycetous fungus, has been traditionally used for red rice preparation using solid-state fermentation. The objective of this study was to develop an improved pigment-producing strain of M. purpureus MTCC 1090 through genome shuffling followed by detailed analytical estimations of pigments and other bioactive compounds produced by the fusant. Protoplast formation was optimum with 12 h-old mycelia incubated at 30 °C with cellulase, lyticase, and chitinase (40:1:1) for 5 h. Four UV-induced mutants that produced 13.1–39.5% higher amount of yellow, orange, and red pigments in fermented low-grade (cheap) broken rice were used as parents for genome shuffling. After the first round of fusion, four fusants with 35.9–60.52% higher pigment production capabilities were fused again, and finally the fusant F2-19 with distinct culture characteristic was selected under multi-selection pressure. It consistently produced 67%, 70%, and 76% higher content of yellow, orange, and red pigments respectively as compared to the wild-type. High-performance liquid chromatography (HPLC) analysis also reveals clear variation in pigment productions between wild-type and the fusant. Furthermore, HPLC analysis of F2-19 fermented rice extract confirms the production of 186 ± 8.71 and 3810 ± 29.81 mg/kg mevinolin and gamma-aminobutyric acid respectively. Citrinin was not detected. F2-19 fermented rice also has high antioxidant activity (7.92 ± 0.32 mg/g trolox equivalent), with good amount of phenolics (18.0 ± 0.95 mg/g gallic acid equivalent) and flavonoids (2.7 ± 0.26 mg/g quercetin equivalent). Thus, genome shuffling was successfully implemented on M. purpureus for the first time to develop a citrinin-free, better-performing fusant that holds future biotechnological potential.

Key points

• Genome shuffling was performed by recursive protoplast fusion in Monascus purpureus.

• The selected fusant, F2-19, was used in solid-state fermentation using low-grade rice.

• It produced 67–76% higher content of yellow, orange, and red pigments than the wild-type.

• HPLC detected 186 mg/kg mevinolin and 3810 mg/kg γ-aminobutyric acid, but no citrinin.

• F2-19 shows high antioxidant activity with good amount of phenolics and flavonoids.

中文翻译：

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基因组改组改善了红曲霉色素和其他生物活性化合物的生产

摘要

传统上，红曲霉（紫曲霉）是一种产生色素的子囊菌，用于通过固态发酵制备红米。这项研究的目的是开发一种改良的紫癜支原体产生色素的菌株MTCC 1090通过基因组改组，然后对融合剂产生的色素和其他生物活性化合物进行详细的分析评估。原生质体的形成是最佳的，将12小时的菌丝体与纤维素酶，裂解酶和几丁质酶（40：1：1）在30°C孵育5小时。四个紫外线诱导的突变体在发酵的低级（便宜）破碎稻中产生了黄色，橙色和红色色素增加了13.1–39.5％的数量，被用作基因组改组的亲本。在第一轮融合之后，再次融合了4个具有35.9–60.52％的颜料生产能力的融合剂，最后在多选压力下选择了具有独特培养特性的融合剂F2-19。与野生型相比，其黄色，橙色和红色颜料含量分别稳定提高了67％，70％和76％。高效液相色谱（HPLC）分析还揭示了野生型和融合剂之间色素产量的明显差异。此外，F2-19发酵大米提取物的HPLC分析证实，分别产生了186±8.71和3810±29.81 mg / kg的甲萘灵和γ-氨基丁酸。未检测到Citrinin。F2-19发酵米还具有很高的抗氧化活性（当量trolox当量为7.92±0.32 mg / g），酚类（当量没食子酸当量为18.0±0.95 mg / g）和类黄酮（当量槲皮素当量为2.7±0.26 mg / g）。因此，基因组改组成功地在 分别为81 mg / kg甲羟萘林和γ-氨基丁酸。未检测到Citrinin。F2-19发酵米还具有很高的抗氧化活性（当量trolox当量为7.92±0.32 mg / g），酚类（当量没食子酸当量为18.0±0.95 mg / g）和类黄酮（当量槲皮素当量为2.7±0.26 mg / g）。因此，基因组改组成功地在 分别为81 mg / kg甲羟萘林和γ-氨基丁酸。未检测到Citrinin。F2-19发酵米还具有很高的抗氧化活性（当量trolox当量为7.92±0.32 mg / g），酚类（当量没食子酸当量为18.0±0.95 mg / g）和类黄酮（当量槲皮素当量为2.7±0.26 mg / g）。因此，基因组改组成功地在紫癜分枝杆菌首次开发出无citrinin，性能更好的融合剂，具有未来生物技术的潜力。

关键点

•基因组改组是通过紫杉菌的递归原生质体融合进行的。

•所选的融合剂F2-19用于使用低级水稻的固态发酵中。

•它产生的黄色，橙色和红色颜料含量比野生型高67-76％。

•HPLC检测到186 mg / kg的甲萘灵和3810 mg / kg的γ-氨基丁酸，但未检测到桔皮素。

•F2-19具有很高的抗氧化活性，并带有大量的酚类和类黄酮。