**1. Introduction**

Quinoa (*Chenopodium quinoa* Willd.), also known as quinoa flour or grey rice. Its seeds are rich in high-quality protein and have high nutritional value [1]. In particular, the growing popularity of quinoa seeds is due to the rich content of proteins, dietary fibers, B vitamins, and dietary minerals as well as for being a gluten-free food [2]. It is a perfect "whole food" for humans and is listed as one of the top ten healthy nutritional foods globally [3]. Quinoa protein isolate (QPI) is widely used in the food industry for preparation of infant foods, edible films, beverages, sauces, and sausages due to its glutenfree nature [4]. In addition, quinoin (a type 1 ribosome-inactivating protein) is contained in quinoa seeds. Quinoin is considered a toxic protein present in both quinoa seeds and sprouts, which is resistant to both heat treatment and in vitro digestion [5]. Therefore, it is necessary to treat quinoa seeds by heating before consumption.

The traditional method of quinoa protein extraction mainly involves alkaline solubilisation and acid precipitation. However, due to disadvantages such as low extraction rate, long extraction time, and ability to change the structure and properties of the active material, the application of this traditional method for QPI extraction is limited. Ruiz et al. [6] demonstrated that the thermal stability of QPI worsened with an increase in pH at the time extraction. Guerreo-Ochoa et al. [7] reported that the yield of QPI obtained by the traditional alkaline extraction method was only 62.1%. Literature suggests that ultrasound, due

**Citation:** He, X.; Wang, B.; Zhao, B.; Yang, F. Ultrasonic Assisted Extraction of Quinoa (*Chenopodium quinoa* Willd.) Protein and Effect of Heat Treatment on Its In Vitro Digestion Characteristics. *Foods* **2022**, *11*, 771. https://doi.org/10.3390/ foods11050771

Academic Editor: Filipa V. M. Silva

Received: 8 February 2022 Accepted: 3 March 2022 Published: 7 March 2022

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to its cavitation properties, can shorten the extraction time and increase the extraction rate of proteins [8]. Additionally, improvement in solubility was reported for rice protein extracted by ultrasound-assisted alkaline extraction method [9]. The optimal ultrasound-assisted extraction conditions for rice bran protein were: amplitude 76%, extraction time 18 min, solid-liquid ratio 0.99 g/10 mL; and the protein yield was 4.73 ± 0.03% [10]. Moreover, in comparison with the traditional extraction method, ultrasound-assisted method increased the protein yield and protein content of duck liver by 67.7% and 4.6%, respectively [8].

The structural and functional properties of proteins play an important role in food processing and determine the application range of proteins. Therefore, research on the effects of different treatments on the structural and functional properties of food proteins is essential to better understand their roles in food systems. Various treatments such as heat [11], ultrasound [12], high pressure [13], and pH [14] significantly affect the structural and functional properties of proteins, among which the heat treatment has been one of the major methods to modify the characteristics of food ingredients and has a great impact on the structural and functional properties of proteins [15]. What's more, most food proteins undergo structural transformation during heat processing which could affect the digestion of proteins [16]. The heat treatment reduced the intermolecular forces of soluble proteins and increased flexibility [17]. Protein from Pacific oyster showed higher digestibility at relatively low temperature, but lower digestibility at relatively high temperature [18].

Although previous studies have documented the effects of heat treatment on the in vitro digestion characteristics of pea protein [19], whey protein [20], and soybean protein [21], there are few references about studies on the optimisation of ultrasounic-assisted alkaline extraction of QPI and the effect of heat treatment on the in vitro digestion characteristics of QPI. Therefore, this study aimed to (1) determine the optimum process conditions for ultrasound-assisted alkaline extraction of quinoa protein; (2) compare the optimum process conditions, extraction rate, and purity of QPI obtained by ultrasound-assisted alkaline extraction process and alkali-solution and acid-isolation extraction process; (3) investigate the effect of different heat treatment time and temperature on structural and functional properties of QPI; and (4) determine the effect of heat treatment on its in vitro digestion characteristics.

#### **2. Materials and Methods**

#### *2.1. Materials*

Quinoa was planted in the open field at Wanggeertang town (East longitude 102.847758, North latitude 35.216012, Altitude 2500 m), Xia he County, Gan nan Tibetan Autonomous Prefecture, China in May. Commercial mature quinoa was harvested in September of the same year. Within 3 h of mechanical shelling, quinoa was packed in woven bags and transported to the laboratory. Samples were stored at 4 ◦C for later use. Pepsin from porcine gastric mucosa (power, ≥250 units/mg solid) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Pepsin (Potency: 1:3000) was purchased from Shanghai Yuanye Biological Technology Co., Ltd., (Shanghai, China). The rest of the chemicals used are of analytical grade.

#### *2.2. Extraction of Quinoa Proteins*

Extraction of quinoa proteins was carried out as described by Silventoinen, & Sozer [22]. The quinoa seeds was crushed, sifted using a 60-mesh sieve, and degreased with petroleum ether (Auto fat analyzer, Sex 406, Jinan Haineng Instrument Co., Ltd., Jinan, China). The degreased quinoa flour was air-dried followed by addition of distilled water to obtain a certain solid-liquid ratio. The pH was adjusted using 1 M NaOH followed by extraction of quinoa proteins at a set ultrasonic temperature. The extract was then centrifuged for 15 min at 6369× *g* (H−1850R, Changsha Xiangyi Centrifuge Co., Ltd., Changsha, China). And the supernatant was collected. The pH of the supernatant was adjusted to isoelectric point of QPI using 1 M HCl and incubated at 4 ◦C for 120 min. After that, the extract was centrifuged for 15 min at 6369× *g* to collect the precipitate. The precipitate was resuspended in distilled water and washed 3–5 times. The pH was adjusted to 7.0 using 1 M NaOH. QPI was obtained by vacuum freeze drying the resuspended precipitate in a vacuum freeze-drying machine (LyoQuest-85, Telstar Lab, Barcelona, Spain) and stored at −20 ◦C until use.
