Gluten-free pasta elaborated with taro flour (Colocasia esculenta): a study of the employ of egg white and transglutaminase on the technological properties

Taro (Colocasia esculenta), like any tuber, lacks the proteins that form the gluten network, making it a possible source of carbohydrates in the production of gluten-free products. This study aimed to enable the production of gluten-free pasta elaborated with taro flour. To help with the pasta structure, egg white was used as a protein source, and the transglutaminase enzyme was used as a processing aid. Pasta was produced with varying the contents of egg white powder (5 to 50% replacement in flour) and transglutaminase (0.005 to 0.05% flour base) according to a complete 22 factorial design containing 7 assays. The pasta was evaluated for pH, instrumental color parameters, cooking properties, and texture. The results were analyzed using Response Surface Methodology. Egg white contributed to increasing the chroma and hue angle, while transglutaminase only increased the chroma. Both egg white and transglutaminase showed a positive interaction effect for cooking time and increased weight of cooked pasta, and a negative interaction effect on firmness. However, these raw materials had no significant effect on the volume increase of the cooked pasta and cooking loss. The lowest contents of egg white and transglutaminase enzyme used in this study have already sufficient to structure the gluten-free pasta elaborated with taro flour, which provides adequate technological characteristics to it.


Introduction
About 10-15% of the world population has some food-related disorder related to cereal proteins. The most known are wheat allergy, celiac disease, and non-celiac gluten sensibility (O'Bryan et al., 2012). The only effective treatment for such disorders is a diet that restricts these proteins. Some of this treatment's problems are the cost and availability of foods free from cereals proteins and its eventual nutritional deficiency (Ciantelli et al., 2012;Figueira et al., 2011). The development of new products using raw materials with added nutritional value is essential in improving individuals' nutritional aspects with foodrelated disorders to cereal proteins (Tomicki et al., 2015). Taro, being a tuber, lacks the proteins that form the gluten network.
It has a high nutritional and energetic value, a rich source of carbohydrates, vitamins, and minerals, besides resistant starch and mucilage. Therefore, it becomes a possible source of nutritionally essential compounds in the production of gluten-free products. Besides, taro is an exciting matrix from a technological point of view, as it has a large amount of starch in its composition, around 70-80% on a dry basis (Kaushal et al., 2015). It is known that starch and proteins form the basis for structuring pasta (Larrosa et al., 2016). In comparison with wheat, used in traditional pasta, taro has a low protein content (taro = around 2% dry basis x wheat = 9 -16% "as is" weight of whole grain), in addition to presenting much smaller starch granules (taro = 0.25 -0.5 μm x wheat = 1 -45 μm) (Bekes & Wrigley, 2004;Kaushal et al., 2015;Lawton, 2004). Thus, to structure a pasta containing taro as a single source of starch, it would be necessary to insert in the formulation a protein resource that could form a network to retain the small starch granules of the taro in the structure, thus avoiding the cooking loss.
Egg white is an interesting protein raw material because, it was found that its inclusion in the formulation of glutenfree bread could increase the cohesiveness and elasticity of the dough during the process, improve color and taste perception due to Maillard Reaction, in addition to reducing their glycemic index (Stantiall & Serventi, 2017). Egg white proteins greatly benefit human nutrition because they have a high content of essential amino acids, which reach 385 amino acids only in ovalbumin, predominantly containing lysine and sulfur (Seuss-Baum et al., 2011). Among the proteins, the main ones can be highlighted: ovalbumin, ovotransferrin, ovomucoid, ovomucin, lysozyme, and globulin (Strixner & Kulozik, 2011).
The insertion of transglutaminase into the gluten free pasta could also help in product structuring as it catalyzes the formation of intra and intermolecular cross-linking in proteins through a transfer mechanism of an acyl group involving proteins that have glutamine residues and primary amines, including the ε-amino group of the lysine residues of certain proteins (Steffen et al., 2017;Susanna & Prabhasankar, 2015). Egg white proteins have been shown to be a suitable substrate for the action of this enzyme in studies of gluten-free bread (Smerdel et al., 2012;Onyango et al., 2010), gels (Alavi et al., 2020) and edible films (Peng et al., 2017).
This study aimed to make feasible gluten-free pasta production made with taro flour, verifying whether the egg white powder and the enzyme transglutaminase could act as product structuring agents.

Material and Methods
A hypothetical scientific method and quantitative assessment to collect data were used in this study as described by Pereira et al. (2018).

Taro flour production
The rhizomes were selected, washed in tap water, peeled, and sliced with a stainless peeler (Vitorinox, Brazil). The slices, approximately 2 mm thickness, were placed on a stainless steel baking sheet and taken to dry in a turbo oven (FTT150, Tedesco, Brazil) with forced ventilation at 50°C and open exhaust for 3.5 h. The dried slices were milled using a grinder (Metvisa, Brazil) until obtaining particles lower than 28 mesh. The flour obtained was stored in polyethylene bags and kept in a freezer at -12 ± 2 °C.

Characterization of raw materials
Taro flour and egg white powder (Maxxi Eggs, Brazil) were characterized in terms of moisture content, pH and water absorption and water solubility indexes (WAI and WSI) according to methods 44-16.01 and 02-52.01 of AACC Approved Methods of Analysis, and Almeida et al. (2010), respectively.

Pasta production
For the pasta production, the flour was considered a mixture of taro flour and egg white powder. The egg white powder content in this mixture varied from 5 to 50% in the flour. The transglutaminase content (Alphamalt PT21006, Mühlenchemie, Germany) ranged from 0.005 to 0.050% flour basis according to a complete 2² factorial experimental design containing 7 assays (Table 1). The amount of water in tests 5, 6, and 7 was determined experimentally. For the other tests, it was calculated proportionally considering the moisture content and water absorption index of the proportion of raw materials present in the flour's composition. Research, Society and Development, v. 10, n. 1, e52710111454, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i1.11454 with the exhaust open and the fan on. The pasta was packed in polyethylene packaging and stored at room temperature.

pH
The pH was determined according to method 02-52.01 of AACC Approved Methods of Analysis.

Instrumental color
Instrumental color of uncooked pasta was determined in CR-400 Chroma Meter (Konica Minolta, Japan) with 10° observation angle, illuminant D65, and using CIEL*C*h color space.

Cooking test
The cooking properties of the pasta were evaluated according to method 66-50.01 of AACC Approved Methods of Analysis with slight adaptations. Portions (6 g) of pasta were added to 100 mL beakers containing 60 mL of distilled water at 100°C. Beakers were partially capped to reduce evaporation and maintain a constant temperature. Every 30 s a pasta piece was removed and pressed between two glass plates until the optimum cooking time was observed. The experiment was repeated until the cooking time but without taking out samples, to make the other evaluations. After the cooking, pasta was washed with distilled water and then drained for 1 min. Cooking loss was carried out by placing the cooking and rinsing water in previously tared crucibles to evaporation until constant weight in an oven at 100°C. It was calculated by using the Eq. 1. The weight increase was calculated by difference between weights of cooked and uncooked pasta according to Eq. 2. To determine the volume increase, the uncooked pasta and cooked pasta were placed separately in one 25 mL graduated cylinder containing 15 mL of petroleum ether and the volume displaced was measured. It was calculated by using the Eq. 3.

Firmness
The pasta's firmness at the optimum cooking time was performed using a CT3 Texture Analyzer version 2.1 "load range" 4.5 kg (Brooksfield, USA) with the aid of TexturePro CT software. The evaluation was carried out by compressing the TA7 (knife-edge) probe into 5 cooked samples placed parallel to each other, perpendicular to the probe. The compression was 100% to the initial height, with a pre and post-test speed of 2 mm/s and a test speed of 0.2 mm/s.

Statistical analysis
All analyzes were performed in triplicate, except for texture analysis, which was performed using eight replicates. The

Characterization of raw materials
The moisture content and pH values found for taro flour were expected (Table 2). They are close to that found by Rodríguez-Miranda et al. (2011): 6.22 ± 0.06 % and 6,78, respectively. The pH for egg white is also according to expected.
The pH of egg white from a newly laid egg is 7,6-8,5, and it increases until 9,7 throughout storage (Stadelman & Cotterill, 2013). The pH of these raw materials is extremely important because they will make up most of the pasta formulation. In this way, they will interfere in the pH of the pasta and, consequently, in the enzymatic activity of the transglutaminase (Damodaran & Parkin, 2017).  (2021) Taro flour had a higher water absorption index, and lower water solubility index than egg white powder (Table 2).
These differences are directly related to the inherent composition of these raw materials. Egg white is predominantly made up of proteins, while carbohydrates, lipids and minerals are minor components (Mine, 2008). Taro has a high content of carbohydrates, fibers and minerals and a low content of proteins and lipids (Kaushal et al., 2015). Carbohydrates and proteins in the taro combine to form a water soluble gum or mucilage, which has considerable thickener and stabilizer potential (Njitang et al., 2014). The presence of this mucilage in the taro was possibly responsible for the considerable WAI presented.
WAI e WSI can be used to predict how materials will behave in a future process. WAI reflects the amount of water bound by the product, while WSI reflects the amount of small molecules solubilized in water (Oikonomou & Krokida, 2011). The WAI value was taken into account along with the moisture content value of raw materials to calculate the amount of water to be added to the pasta. The results are showed in Table 1. Thus, it is observed that the higher the content of taro flour in the flour, the greater the amount of water added to the pasta formulation.

pH
The pasta's pH value varied from 6.35 ± 0.01 to 7.99 ± 0.01 (Table 3). The egg white had a positive effect in this parameter, as observed in the mathematical model and response surface obtained for this parameter. The increase of egg white content promoted an increase in the pH value of the pasta (Figure 1). No influence on the pH of pasta was observed with the variation of transglutaminase content. pH is among the most important environmental factors the influence enzyme activity, and change this parameter compromise one of the principal physical means to control enzyme action in food matrices (Damodaran & Parkin, 2017). In this way, the increase in the egg white content in the formulation will affect the activity of transglutaminase by affecting the pH of the pasta. According to the enzyme manufacturer (Mühlenchemie, 2013), the transglutaminase employed in this study has an optimum pH between 6.0-7.0. This pH range was obtained in the pasta elaborated with egg white content between 5-25% ( Figure 1).  In the models: EW = coded value of egg white (-1 to 1); TG = coded value of transglutaminase (-1 to 1) Source: Authors (2021)

Instrumental color
Through the results of the instrumental color parameters evaluated (Table 3), it is observed that the uncooked pasta had lightness (L*) between 73.41 and 76.26, chroma (C*) between 16.20 and 19.74, and hue angle (h) between 75.50° and 83.73°, that is, pasta presented a very pale yellowish color according to Konica Minolta (2015), which can be observed in Figure 2. The pasta color is an essential criterion for assessing the quality of the pasta as it is the first characteristic noticed by consumers, which leads to the immediate acceptance or rejection of the product (Bouasla et al., 2017;Marti et al., 2013). The ideal conventional pasta should present a uniform pale yellow color without cracks (Rosarlo et al., 1999;Sicignano et al., 2015).  Table 1.
The increase in the egg white content contributed to an increase in the yellowish hue (h) of the pasta and increase the color saturation (C*), as can be observed in the mathematical models and response surfaces obtained for these parameters ( Figure 1). The egg white powder presents a dark yellow color, which is developed during its hot-air drying process. The egg white has glucose (reducing sugar) and proteins with an amino group, that allows the occurrence of the Maillard Reaction (Katekhong & Charoenrein, 2017).
Transglutaminase had no significant effect on the hue angle but also increased color saturation (C*). With the increase in the enzyme content, there is an increase in the polymerization of the egg white proteins (Alavi et al., 2020), which can alter the reflection/refraction of light. Higher values of chroma were found when higher levels of egg white and transglutaminase were used together.
Egg white and transglutaminase had no significant effect on the pasta lightness (Table 4); regardless of the variation in the contents of these raw materials, the lightness value was within an average value standard deviation that was 74.69 ± 1.09.

Cooking test
Through the analysis of the results, it was possible to obtain mathematical models that explain how variations in the egg white and transglutaminase contents influenced the variation in cooking time and weight increase of cooked pasta ( Figure   3). Egg white and transglutaminase contributed positively to the increase in cooking time and weight increase, showing a positive interaction effect in these two parameters. The cooking time between 7.3 and 44.3 min was obtained in the assays; that is, there was a significant variation in this parameter (Table 5). Very long cooking times are not feasible both from an energy point of view and from a practical point of view. These very long periods were obtained when we used the highest contents of egg white and transglutaminase together. In conditions where we used low levels of transglutaminase and high levels of egg white simultaneous and, in high levels of transglutaminase and low levels of egg white simultaneous, we observed that the cooking time remained at the lowest values found in the study. Therefore, the isolated increase of only one of these ingredients was not as impactful in the cooking time as the increase in these two variables together. This demonstrates the possible role of transglutaminase on the amino acids of egg white proteins because we verified these variables' interdependence. We know that for an enzyme to express its activity, the presence of its substrate is necessary. Transglutaminase cross-links with protein molecules through covalent bonds formed between ɛ-amino and γ-carboxamide, resulting in isopeptide bonds 20 times stronger than non-covalent bonds (Alavi et al., 2020). As Yao et al. (2020) pointed out, pasta with a higher protein content tends to have a more compact structure that makes it difficult for water to enter, increasing its cooking time (Wang et al., 2011).  Regarding the weight increase, these same findings are valid, which can be observed by the similarity of the response surfaces obtained (Figure 3). Pasta of the experimental assays showed 144.11 to 234.23% weight increase during cooking (Table 5). The action of transglutaminase on egg white proteins probably allowed the formation of a more interconnected protein network that increased the product's water absorption capacity, which reflected in a weight increase.
It was not possible to verify a significant effect of egg white and transglutaminase on the volume increase of cooked pasta and cooking loss (Table 4); that is, regardless of the variation in the contents of these raw materials, the volume increase and cooking loss values were within an average value and its standard deviation, which were 24.9 ± 8.6% and 9.12 ± 0.79%, respectively.

Firmness
Regarding firmness, higher values were found in formulations with higher contents of egg white and lower contents of transglutaminase (Figure 3). At medium and higher levels of egg white, the increase of transglutaminase made the product softer. This effect of the enzyme was already verified in a previous study using protein gels. Sakamoto et al. (1994) found that transglutaminase's addition to a specific concentration increased the gel's firmness. However, in excess, the enzyme made the gels soft and fragile. These authors believe that the excessive formation of cross-links between glutamine and lysine could inhibit the protein network's uniform development.

Conclusion
The production of gluten-free pasta elaborated with taro flour was possible through the insertion of egg white and transglutaminase as structuring agents. The lower levels of egg white and transglutaminase used in the study (5% flour replacement in flour and 0.005% flour base, respectively) have already allowed pasta to have good color, cooking properties, and texture, which shows good performance from a technological point of view. Further studies are needed to investigate the sensory perception, nutritional value and functional properties attributed to the gluten-free pasta elaborated with taro.