An effective method for extracting anthocyanins from blueberry based on freeze-ultrasonic thawing technology
Introduction
Blueberries (Vaccinium spp.) are known as a “superfood” due to their unique flavor, health benefits, and high nutritional value [1]. Studies have demonstrated potent antioxidant [2], anti-cancer [3], anti-inflammatory [4] and cardioprotective [5] effects of blueberries, which can be attributed to its bioactive compounds, including anthocyanins, flavonols, phenolic acid, various vitamins and minerals [6]. Anthocyanins are a class of antioxidant flavanoids that are abundant in berries, grapes and other dark-colored fruits. They are natural phytopigments that impart dark purple, blue or red colors to the plants. Blueberries have the highest content of anthocyanins, and are therefore recommended as a functional food as well as dietary supplement. Although synthetic anthocyanins have been widely used in the food industry in recent years, the natural compound is still preferred, calling for more effective methods of extraction anthocyanins from different sources [7].
Solvent extraction, ultrasonic-assisted extraction (UAE), enzyme-aided extraction (EAE) and other methods have been used to extract anthocyanins from blueberry Vaccinium spp. (ABVS) [8], [9], but are limited by the poor yield and quality of the extract, and potentially harmful byproducts. For example, solvent extraction process requires an acidifier that hydrolyzes acylated anthocyanins. Furthermore, the organic solvent may have toxic side effects [10]. UAE is an efficient, economical and eco-friendly method of extracting anthocyanin. The bubbles produced by cavitation effect accelerate plant cell disruption and increase extraction yield [11]. However, the high ultrasonic frequency and high temperature can destroy the structure of anthocyanins. Although a lower ultrasonic frequency can increase the stability of anthocyanins, it prolongs the extraction process. Finally, enzyme extraction requires a highly specific enzyme, and only a few can be used to extract anthocyanin from blueberries [12]. Therefore, it is necessary to develop a novel method for rapidly and efficiently extracting anthocyanins from blueberries for biomedical applications.
While the influences of freeze-thawing on food and plants have been of interest to researchers [13], [14], to the best of our knowledge, the freeze-ultrasound thawing extraction (FUTE) of anthocyanins from blueberry has not previously been reported. The ice crystals formed during freezing fruits and vegetables rupture the cell walls and parenchyma [15], which adversely affects their texture and consistency during the subsequent thawing. At the ultrastructural level, repeated freeze–thaw cycles can solubilize protein aggregates, and release vacuolar contents and cellular inclusion bodies. Therefore, freeze-thawing is also a viable strategy to extract anthocyanins and other plant pigments. However, prolonged thawing of frozen blueberries at room temperature may reduce the yield of anthocyanins. Then, UAE is another common method used to extract anthocyanins, because it is efficient, economical, and environment friendly [16]. We hypothesized that combining freeze-thawing extraction (FTE) and UAE could lower the temperature of ultrasonic thawing and shorten the extraction time, which in turn can enhance both the yield and stability of the extracted ABVS. (Fig. 1).
We developed a freeze-ultrasonic thawing technology (FUTE) to rapidly and efficiently extract ABVS. We optimized the parameters of FUTE, including freezing time, ultrasonic thawing time, ultrasonic thawing temperature and liquid-solid ratio by single-factor design and multiple response surface methodology, with anthocyanin and cyanidin-3-O-glucoside yield as the responses. The yield and antioxidant capacity of ABVS extracted by FTUE, UAE and FTE were compared to determine the efficacy of FTUE.
Section snippets
Plant material
Blueberry (Vaccinium spp.) samples were purchased from Sichuan Chaoyue Agricultural Science and Technology Co. Ltd. (Sichuan, China), and identified by Dr. Tingting Xu from Huaiyin Institute of Technology, Huai’an, Jiangsu, PR China. A representative specimen was deposited in the Key Laboratory of Regional Resource Exploitation and Medicinal Research of Huaiyin Institute of Technology. Samples were stored at −4 °C, and only intact frozen fruits with blue skin color and uniform size were
Selection of extraction solvent
The extraction solvent is a crucial factor to consider for optimizing product yield. Studies show that ethanol is the most effective solvent for extracting anthocyanins from different plant materials [11]. We tested water and ethanol by a one-factor-at-a-time (OFAT) approach, and examined the epiderma cells on the blueberry peels after extraction. As shown in Fig. 2, anthocyanin was more or less retained in the blueberry epidermis following water extraction (Fig. 2A), but completely leached by
Conclusion
FUTE is a rapid and effective method for extracting anthocyanins from blueberries. The optimum conditions of FUTE as per RSM were freezing time 5.43 min, ultrasonic thawing time 23.56 min, ultrasonic thawing temperature 41.64 °C, and liquid–solid ratio 24.07:1 mL/g. FUTE was also superior to UAE and FTE in terms of the yield and antioxidant function of the extracted anthocyanins. This novel technique can used to extract anthocyanins from other plants as well.
CRediT authorship contribution statement
Jun Yuan: Conceptualization, Methodology, Writing - original draft. Hailun Li: Data curation, Writing - original draft. Weili Tao: Visualization, Investigation. Qian Han: Visualization, Investigation. Huiqing Dong: Visualization, Investigation. Jin Zhang: Visualization, Investigation. Yi Jing: Software, Visualization. Yanming Wang: Software, Visualization. Qingping Xiong: Supervision. Tingting Xu: Supervision.
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.
Acknowledgments
This work was partly supported by Six Talent Peaks Project in Jiangsu Province, China (2017-YY-003), Scientific Research Project from Jiangsu Commission of Health, China (H2019062) and Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (HGYK201901).
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These authors contributed equally to this work.