Improvement of flavonoid aglycone and biological activity of mulberry leaves by solid-state fermentation

https://doi.org/10.1016/j.indcrop.2020.112287Get rights and content

Highlights

  • Solid-state fermentation was applied for mulberry leaves with Monascus anka.

  • The dynamic changes in enzymes activities and phenolic contents were investigated.

  • Fermented mulberry leaves showed higher biological activities than untreated leaves.

  • Fermentation of mulberry leaves was as a new strategy for the production of quercetin and kaempferol.

Abstract

Mulberry leaves contain various types of phenolics, especially glycosides of the flavonoids quercetin and kaempferol. The aim of the present study was to investigate the production of quercetin and kaempferol as well as the biological activity of mulberry leaves with solid-state fermentation by edible fungi. Among the test fungi, the fermentation with Monascus anka was found to obviously increase the level of quercetin and kaempferol in the mulberry leaves. After fermentation, the amounts of quercetin and kaempferol were up to 5.30 ± 0.35 mg/g dw and 1.87 ± 0.10 mg/ dw, respectively. Meanwhile, the changes in enzymes activities and the contents of quercetin and kaempferol during fermentation were systematically studied. The morphology observation demonstrated that the mulberry leaves were severely damaged after fermentation. In addition, the biological activities including free radical scavenging activity, α-glucosidase inhibition activity, and antimicrobial activity significantly enhanced with fermentation. This study provided a scientific basis for the production of the high value flavonoid aglycones from plant materials.

Introduction

Mulberry [Morus alba L.] is a perennial woody plant and widely distributed all over the world (Sánchez-Salcedo et al., 2016). The leaves of mulberry, as the best feed for silkworms (Bombyx mori L.), are well known in the silk production. In China, mulberry played a big role in the progress of China silk road for centuries. Moreover, mulberry leaves are also a good source of polysaccharides, phenolic acids, and flavanol derivatives which exhibit positively impact on human health. It has been reported that the mulberry leaves can be taken as the traditional herbal medicines, as well as be processed into various functional foods (Li et al., 2017; Sheng et al., 2018). With the advantages of the fast growth and short proliferation period of mulberry trees, the annual output of mulberry leaves can reach 20 tons per hectare. However, only 1–3 % of mulberry leaves resource can be utilized (Cai et al., 2019). Therefore, it is of immeasurable importance to exploit the value of mulberry resources.

The plant flavonoids are regarded as the most important polyphenols in human life due to their diverse biological activity (Xiao, 2017). Among flavonoids, the flavonoid aglycones and flavonoid glycosides have received more widespread attention than others. Compared with flavonoid glycosides, flavonoid aglycones have been reported to enhance the nutrient absorption efficiency in the human intestine better than its derivatives (Li et al., 2009). Moreover, flavonoid aglycones exhibit stronger bioactivities, such as antioxidant activity and antidiabetic activity, etc. Quercetin and kaempferol have been proved to possess higher antioxidant potential than their glycosides in ABTS assay, DPPH assay, and other antioxidant systems (Cai et al., 2006; Burda and Oleszek, 2001). The glucoamylase inhibition activity of quercetin were greater than any other derivatives, such as isoquercitrin and rutin (Qin et al., 2013). Considering the special effects of flavonoid aglycones on people health, the development of plant flavonoid aglycones, especially frequently mentioned quercetin and kaempferol, are urgent to broaden and improve the nutritional or medicinal values of plant resources.

Nowadays, some carbohydrate-hydrolysing enzymes such as cellulase, xylanase, and β-glucosidase have played an important role in breaking the cell walls, which can lead to the dissociation of phenolics and the deglycosylation of glycosides to aglycones (Neri et al., 2009; Jin et al., 2013). However, because the high cost associated to the production of commercial enzymes restricts large-scale applications, in recent years, there has been a lot of interest in using the fermentation technique as an alternative for the production of enzymes. Both submerged fermentation (SmF) and solid-state fermentation (SSF) have been employed for microbial enzymes production. Compared with SmF, SSF has been applied in plant chemistry, given the advantages of consecutiveness selectivity, simple operation, moderate cost and environmental friendliness (Foong et al., 2009). It has been used in Psidium guajava L. leaves, cauliflower outer leaves, oats and other plants (Wang et al., 2016; Huynh et al., 2016; Bei et al., 2017).

It is well-known that the mulberry leaves are rich in the quercetin and kaempferol glycosides. The mulberry leaves therefore can be taken as a good source to produce quercetin and kaempferol aglycones. Considering the food safety of microorganisms, the aim of this study was to develop an SSF method in mulberry leaves to enrich the quercetin and kaempferol with edible fungi strains. The enzymes and the fingerprint of phenolic compounds in mulberry leaves were dynamically investigated. Subsequently, the bioactivities of leaves before and after fermentation were compared. The result would be of considerable significance to provide a feasible strategy for the production of flavonoid aglycones from the abundant plant resources.

Section snippets

Materials and reagents

The edible strains of Yeast CICC 1912, Aspergillus oryzae 3.591, Monascus anka 3.782 and Aspergillus niger 3.3883 from the Heilongjiang Institute of Microbiology (China) were used. The cultures were stored on potato dextrose agar (PDA) at 4 °C before use. Mulberry leaves were obtained from Zhangjiajie (China). They were air dried, then powdered by a disintegrator and sieved (40 mesh). The standards of quercetin and kaempferol, rutin, isoquercitrin and astragalin (HPLC ≥ 98 %) were bought from

Selection of microorganism for quercetin and kaempferol production

The edible fungi, which can produce beneficial metabolic products, has been widely used in fermented food. Moreover, the significant amounts of carbohydrate-hydrolysing enzymes (especially cellulase and β-glucosidase) from edible fungi promoted the release of leaves phenolic constituents and converted them to high value components. Recently, compared with flavonol glycosides, many studies had been focused on the flavonoid aglycones for the apparent gastrointestinal absorption and biological

Conclusions

The present investigation demonstrated that the solid-stated fermentation with Monascus anka was an effective approach to enhance the yield of quercetin and kaempferol from mulberry leaves. The higher contents of quercetin and kaempferol were obtained after ten days fermentation. The morphology observation of the mulberry leaves showed that the cell walls structures were severely damaged by fermentation treatment, which leaded to the dissociation and conversion of flavonoid glycoside to

CRediT authorship contribution statement

Na Guo: Conceptualization, Methodology, Investigation, Writing - original draft. Ya-Wei Zhu: Validation, Formal analysis. Yi-Wei Jiang: Software. Hong-Kun Li: Validation. Zhi-Ming Liu: Writing - review & editing. Wei Wang: Writing - review & editing. Chun-Hua Shan: Writing - review & editing. Yu-Jie Fu: Resources, Writing - review & editing, Supervision, Data curation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge the financial supports by National Key R&D Program of China (2016YFD0600805), the Fundamental Research Funds for the Central Universities (2572017EA03, 2572018CT01, 2572018AB12) and Heilongjiang Touyan Innovation Team Program (Tree Genetics and Breeding Innovation Team).

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