**1. Introduction**

In today's health-based consumer-focused world, more and more research is being conducted in order to obtain knowledge about the effects of animal- and plant-based diets on our health. It has generally been accepted that the consumption of meat can increase the incidence and prevalence of obesity, cardiovascular diseases and stroke, cancer, type 2 diabetes, and patient mortality. Contrarily, the plant-based diet has been accepted to lower the risk of these factors [1,2]. Consequently, an increasing percentage of people are changing their diets to become vegetarians, vegans, or flexitarians in order to lower their meat consumption and increase the amount of vegetables and fruits [3,4]. Yet, false information and health trends, which state that plant-based proteins are worse for your health, inadequate for building muscle, or are not proteinaceous enough, have become powerful and generally accepted misconceptions among some members of the population [5]. Research has managed to prove that legumes can be a good source of protein, though the bioavailability of animal proteins was still proven to be higher [6,7]. The recommended acceptable intake of proteins is around 0.8 g/kg for adults (defined as the average daily level of intake sufficient to meet the nutrient requirements of nearly all healthy people). Legumes have a great potential for delivering quality proteins, but raw legumes have a lower degree of bioavailability and, thereby, lower nutritional value as compared to other foodstuffs. Upon ending the last century, food was used to improve health, while entering the millennium a paradigm shift resulted in that our knowledge is now being used to improve foods in respect of healthiness. Both animal- and plant-based proteins have been widely investigated in order to understand the digestibility as such. Since it is well known that different processing procedures will affect both structure and functionality of proteins, it is anticipated that processing may also affect protein digestibility. This communication surveys the effect of different processing methods on legume protein digestion (PD) in order to contribute to the knowledge of protein digestibility. It is not a comprehensive review of all research studies on PD, but is based on the studies defined by investigations that contribute to provide an overview of the effect of processing on digestion of legume proteins. Thus, to "set the current scene" and give an outline of the challenges and future perspectives.

#### **2. Processing Techniques**

Consumption of raw legumes can be tedious and difficult, and, in worst case, inefficient with respect to amino acid absorption in the gut. Empirical, various domestic processing methods are used to ease the legume consumption per se, but without grasping the effect on protein digestibility as such. Several different types of legumes and of processing techniques have, thus, been investigated in order to evaluate the potential improvement of nutritional value and protein utilization. Processing is the action of performing a series of mechanical or thermal operations on food in order to change or preserve it. It may involve soaking, cooking, microwave irradiation, baking, pressure-cooking, autoclaving, and extrusion [8,9]. Since the processing methods are performed at different conditions, a brief description of the techniques that have been used is presented.

One of the commonly used pre-processing method is soaking. The most important parameters of soaking are: product:water ratio, temperature of the soaking water, and the duration. These parameters differ considerably depending on the type of legume, hence, the legumes are soaked in either hot or boiling tap or distilled water, with legume:water ratios varying from 1:1 to 1:5, and soaking durations ranging from 12 to 16 h. Traditional cooking is the simplest thermal processing method used. In the case of legumes, they are usually either placed in surplus warm water and slowly brought to boiling or directly put into boiling water (100 ◦C) in a pot. The time of the cooking process is a very important factor, which can differ depending on the legume type and if the legume seeds were soaked, dried, or not treated prior to cooking. Nevertheless, in a majority of the reviewed studies, the cooking time varied between 20 and 40 min. Microwave radiation is a more intense and, thereby, faster thermal method than conventional cooking. The thermal effects are related to the heat generated by the absorption of microwave energy by the water in the food matrix. Contrarily to cooking, microwave processing offers the opportunity to provide a more precise amount of energy to the food item. In addition, the heating effect of microwave can be tuned (energy dosage) and is considerably faster, almost immediate, resulting in reduction of cooking time. Similar to cooking, the microwave treatment time varies depending on type of legume and pre-treatment before microwave cooking. The capacity of microwave ovens differs due to different microwave frequency, thus delivering different energy doses, which will influence the treatment duration. In the studies herein, the doses of energy varies from 500 to 2000 J/g, usually being increased by 250 J/g. Pressure-cooking and autoclaving are two other high-intensity thermal methods. They are both based on exceeding normal, ambient boiling point of water in a sealed vessel (pressure cooker) or a pressure chamber (autoclave). The pressure cooker works by trapping the steam produced from boiling water of 121 ◦C inside the vessel. Many autoclaves work by subjecting the item to pressurized saturated steam at 121 ◦C. Thus, they both operate at 1.8 to 2.0 bar in order to obtain this temperature. The evaluated temperature and pressure generally increase reaction rates, thus the duration of pressure cooking and autoclaving is less than traditional cooking, though it varies between the two. Pressure cooking is usually performed in 5 to 15 min, whereas autoclave time may vary from seven to 60 min, both depending on the type and quantity of legume. The last two processing methods are based on milling the legumes into a flour prior to treatment. Baking is an old, traditional thermal method that uses dry air at ambient pressure to apply heat. Generally, a dough based on legume flour is made, rested for some time (leavening), and then baked at temperatures varying from about 180 to 220 ◦C for more than 30 min. Extrusion is also based on transforming legume flour into an edible food product. The extrudates are prepared using either a single- or twin-screw extruder that works under high temperature and high mechanical pressure. The main independent parameters, such as particle size of flour, feed rate, moisture content, barrel temperature, and screw speed, during the extrusion process, differ a lot depending on legume type and extruder parameters (type of mill, screen hole size, etc.). In the reviewed papers, moisture content was about 22–25%, while temperature and screw rotation speed varied considerably from 30 to 150 ◦C and from 100 to 650 rpm, respectively.

#### **3. Protein Digestibility Methodologies**

The major difference between PD assessment is if the measurement is conducted in vivo or in vitro. Furthermore, the digestion of proteins may be determined by a broad variety of methods within these two PD categories. In vivo methods are based on feeding trails using animals or humans. The most accurate result is obtained with controlled feeding experiments with animals, usually rodents, chicken, or pigs are used. However, care should be taken upon transferring results and conclusions from animal tests to human, since the human GI tract differs considerably from animals. On the one hand, human intervention studies are the golden method for assessing protein digestion and nutritional value. On the other hand, in vivo experiments are time consuming and costly, and difficult to control if the study is based on human intervention. Therefore, in vitro models simulating the human GI tract have been used. Contrarily to in vivo methods, in vitro methods are easier to control, rapid to conduct, and may be less expensive. The drawbacks among the in vitro methods are differences in the operation and the experimental parameters; as a result, there are considerable differences in the measured digestibility complicating comparison of results. In addition, the difficulties in accurately transferring and simulating the complex digestion process in the human GI tract complicates the performance of these laboratory experiments and caution must be taken upon interpretation of results. In the following, the various methods used to measure and assess legume protein digestibility in the surveyed papers are summarized.
