Optimization of enzymatic assisted extraction process for anthocyanins from black bean seed coat and analysis of its antioxidant activity
Black beans, also known as black beans, winter beans, and green beans, are the dry and mature black seeds of the legume soybean Glycine max (L.) Merr. They have strong environmental adaptability, drought resistance, and tolerance to barrenness, and can be planted in most places, even in saline alkali land. Black beans have a wide planting area and relatively concentrated regional distribution in China. They are commonly planted in Heilongjiang, Anhui, Shanxi and other places, with abundant resources and diverse types. Black beans are a famous traditional medicinal and edible agricultural product, rich in unsaturated fatty acids, proteins, vitamins, polysaccharides, isoflavones and other bioactive substances. Black bean seed coat is black red or purple red, shiny, thin and brittle, easily broken, and contains various active substances such as dietary fiber, vitamins, pigments, polysaccharides, flavonoids, etc. The anthocyanins in the seed coat of black beans not only endow black beans with color, but also have a variety of health care effects on the human body, such as anti-oxidation, bowel soothing, liver protection, prevention of diabetes, blood sugar, blood lipids, memory improvement, vision protection, anti-tumor, prevention and treatment of asthma, etc.
The gradual improvement of anthocyanin extraction technology has provided a foundation for its industrial application, enabling it to be widely used in industries such as new cosmetics, healthcare, food, and natural dyes. The traditional extraction methods for anthocyanins from black bean skins mainly include water extraction and organic solvent extraction. However, these methods have disadvantages such as long extraction time, high solvent consumption, low extraction rate, and poor stability of anthocyanins. In recent years, methods such as ultrasound assisted extraction, microwave-assisted extraction, and ionic liquid extraction have been explored in the study of anthocyanins in black bean seed coat due to their characteristics of short extraction time and higher extraction rate. Previous studies have found that the enzymatic assisted extraction of bioactive substances such as anthocyanins has the advantages of low energy consumption, high cost-effectiveness, and high stability and activity of the extracted products. However, there have been no reports on the enzymatic assisted extraction of anthocyanins from black bean skins. This study used enzymatic assisted extraction of anthocyanins from black bean skins, and optimized the extraction process of anthocyanins using response surface methodology with anthocyanin content as the indicator. At the same time, in vitro experiments were conducted to analyze the antioxidant activity of the extract of anthocyanins from black bean skins, in order to provide theoretical and technical references for the extraction and comprehensive development and utilization of anthocyanins from black bean skins.

In this experiment, ethanol was used as the extraction solution to optimize the process of extracting anthocyanins from black bean seed coat using a bio enzyme assisted method. The enzymatic assisted extraction of plant active ingredients has the advantages of being green, pollution-free, non-toxic, and harmless. It mainly relies on the fact that most effective active ingredients exist in the cytoplasm of plant cells, and can be biodegraded by cellulases, alpha amylase, and other enzymes on cell wall cellulose, achieving the goal of breaking down the dense structure of the cell wall, enhancing the permeability of the cell wall and intercellular barriers, and maximizing the diffusion and release of effective ingredients from the cell cytoplasm to the extraction medium, thus efficiently extracting bioactive substances.

Based on single factor experiments, the optimal extraction scheme is derived through response surface optimization and validated. The results showed that the response surface model regression was highly significant (P<0.0001), and the mismatch term was not significant (P>0.05). The model can be accurately used to predict the pattern of changes in the content of anthocyanins extracted from black bean skins with various experimental factors. The optimal process conditions obtained through optimization are: composite enzyme (cellulase 400U/g+α – amylase 50U/g), enzymatic hydrolysis temperature of 50 ℃, liquid to material ratio of 26:1mL/g, ethanol volume fraction of 64%, and enzymatic hydrolysis time of 59min. Under these experimental conditions, the extracted anthocyanin content was 2.019mg/g.
Take anthocyanin content of 2.019mg/g. Preliminary studies on the antioxidant activity of anthocyanins in black soybean seed coat indicate that anthocyanins in black soybean seed coat have good antioxidant activity and exhibit a dose-response relationship. Within the experimental range, its reducing ability and ability to scavenge superoxide anion radicals are slightly lower than ascorbic acid, but its ability to scavenge nitrite ions, DPPH radicals, and ABTS radicals is stronger than ascorbic acid, which is consistent with the research results of Xu, Wu, and others. The generation of antioxidant capacity of anthocyanins is due to their conjugated structure, and the stability and reactivity of anthocyanins are influenced by the degree and position of hydroxylation and methylation on the B ring, which in turn affects the antioxidant activity of anthocyanins. Research has shown that cyanidin-3-O-glucoside (C3G) is the main component of anthocyanins in black bean skins, with phenolic hydroxyl groups in its structure. It can serve as a hydroxyl donor, capturing and binding free radicals to form a stable structure, terminating the occurrence of free radical chain reactions, and exhibiting antioxidant capacity.
Experiments have shown that anthocyanins in black bean seed coat have high medicinal value and broad application prospects. However, the enzymatic assisted extraction of natural active ingredients is a complex solid-liquid phase transfer process, and the dissolution of more cellular contents may lead to the simultaneous introduction of more impurities into the crude extract. The content of anthocyanins in black bean seed coat is relatively high, and its crude extract of anthocyanins exhibits certain antioxidant capacity, which has the potential to develop new anthocyanin resources. Anthocyanins are not the only antioxidant substances in black bean seed coat. Therefore, further purification of anthocyanins in black bean seed coat has certain guiding significance for in-depth study of its active ingredients, chemical structure, physicochemical properties, and determination of structure-activity relationship.