**1. Introduction**

Kudzu (*Pueraria lobata*) is a herbaceous, perennial plant native to East Asia and belongs to the Leguminosae family. It is a rich source of starch, dietary fiber, minerals, and isoflavonoids and possesses many nutritional and pharmacological activities [1–3], such as antioxidant, anticancer, anti-inflammation, and neuroprotective properties. Additionally, kudzu is used as a vegetable and edible ingredient in the preparation of various foods [4]. For example, kudzu starch is used in the production of noodles, beverages, oral liquids, and various dishes, and it serves as a sauce thickener [5]. Food products containing components of kudzu have begun to attract interest from both consumers and researchers due to their health benefits. However, enzymatic browning of kudzu during storage, handling, and processing negatively affects its sensory and nutritional properties. Polyphenol oxidase (PPO) is the primary enzyme involved in enzymatic browning [6,7]. Therefore, the inactivation of PPO is important in improving the quality attributes of kudzu products.

Thermal treatment has been widely used as a non-chemical means for enzyme inactivation in food processing. The thermal inactivation of PPO from fruits and vegetables has been well evaluated in mushrooms [8,9], pear [10,11], apple [12,13], chestnut kernel [14], and pineapple [15]. The thermostability of PPO highly depended on the environmental conditions, and an obvious difference was observed in food and model systems. Purified apple PPO was almost completely inactivated after incubation at 65 ◦C for 20 min [13], while PPO in apple slices still retained about 40% relative activity in the same condition [12].

**Citation:** Liu, J.; Zhang, J.; Liao, T.; Zhou, L.; Zou, L.; Liu, Y.; Zhang, L.; Liu, W. Thermal Inactivation Kinetics of Kudzu (*Pueraria lobata*) Polyphenol Oxidase and the Influence of Food Constituents. *Foods* **2021**, *10*, 1320. https://doi.org/10.3390/foods10061320

Academic Editor: Montserrat Dueñas Paton

Received: 14 May 2021 Accepted: 4 June 2021 Published: 8 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Compared to crude PPO extract from pear [11], PPO in pear puree [10] showed higher thermostability at a temperature range from 30 to 100 ◦C. The food system differs from the model system since the complex food constituents may affect the enzymatic properties during food processing [16]. The impact of pectin [17,18], protein [19,20], and sugar [21] on enzymatic properties have been investigated. However, there are few reports comparing the thermostability of PPO in food and model systems in the same study, and the effect of food constituents on the thermal inactivation kinetics remains unclear.

In this study, PPO from kudzu was purified and characterized. The thermal stability and inactivation kinetics of kudzu tissue PPO, crude PPO, and purified PPO were evaluated and compared with each other. To further illustrate the lower thermal inactivation effect in kudzu tissue, the content of food constituents, including pectin, protein, sucrose, and starch, in kudzu was analyzed, and the effect of these constituents on PPO activity, thermal inactivation kinetics, and conformation change was also evaluated.

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

#### *2.1. Materials*

Kudzu root (*Pueraria lobata*) was harvested from the plantation at Shangrao, Jiangxi province in eastern China, and stored at 4 ◦C until processing. Triton X-100, polyvinylpolypyrrolidone (PVPP), and *m*-hydroxydiphenyl were obtained from Aladdin Chemicals Co. (Shanghai, China). Catechol was obtained from Macklin Chemicals Co. (Shanghai, China). Pectin was purchased from Sigma-Aldrich Co. (Shanghai, China). Bovine serum albumin (BSA), starch, sucrose, D-Galacturonic acid, D-Glucose ammonium sulfate, tetramethylethylenediamine (TEMED), phenylmethylsulfonyl fluoride (PMSF), SDS-PAGE loading buffer, and standard protein mixture (Marker) were purchased from Solarbio Science and Technology Co. (Beijing, China). All the other chemicals were of analytical grade. Double-distilled water was used for preparing all the solutions.

#### *2.2. PPO Extraction*

The extraction and purification of PPO from kudzu were carried out according to the method of Liu et al. [22] with modifications. After washing and peeling, kudzu was sliced and mashed using a blender (HX-PB1053, AUX, Zhejiang, China) with 0.1 M phosphate buffer (pH 6.5) containing 2% (*w*/*v*) polyvinylpolypyrrolidone (PVPP), 0.5 M NaCl, and 1% (*v*/*v*) triton X-100 in a proportion of 1:1 (*v*/*v*) to yield puree. The mixture was stirred in an ice-water bath for 2 h and centrifuged (SORVALL Biofuge primo R, Thermo, Germany) for 20 min at 11,000× *g* at 4 ◦C. The supernatant was collected as the crude PPO (cPPO) and stored at 4 ◦C until use.
