Protein solubility of jackfruit seed flour : pH and salt concentration influence

Solubility is a fundamental physicochemical property of proteins because of its importance over other protein properties in foods. Water solubility characteristics determine appropriate protein extraction and fractionation conditions in foods. In this study protein solubility of jackfruit seed flour (Artocarpus integrifolia L.) was determined, combined with the effect of pH and sulfate, chloride and trisodium citrate salt concentration variations. Protein solubility was higher in more acidic (pH =2.0) and alkaline (8.0≤ pH ≥10.0) mediums, in the absence of salts. It was also verified that the best solubility conditions under pH 2.0 using trisodium citrate was at 0.25 mol/L; and for pH 6.0, it was 1.0 mol/L. For sodium chloride, the best conditions occurred at 0.25 mol/L for pH 2.0, 6.0 and 10.0; and the best solubility values using sodium sulfate under pH 4.0 and 8.0 was 0.25 mol/L; and 0.50 mol/L under pH 6.0 and 10.0. Jackfruit seeds flour can be used as an alternative source of edible protein and as a substitute for products already available in the market.


Introduction
Proteins, commonly used as food ingredients, are of fundamental importance for human diets. In addition, they attribute sensorial properties and provide adequate functionality conditions (Waghmare et al., 2019;Zang et al., 2019).
The use of alternative sources of lower cost for substituting or simulating traditional high protein value food relies on the knowledge of functional properties of its protein and its behavior in certain food systems. Recent studies on human nutrition deal with low cost sources of food protein, due to animal's protein high cost and scarce supply, especially in underdeveloped countries. On the other hand, legumes are abundant and they are a cheaper food protein source (Lawal et al., 2007). New compounds or protein sources such as Jackfruit (Artocarpus heterophyllus Lam.), originally from Asia, has acclimated well in Brazil. It is rich in carbohydrates, protein, vitamins, and minerals (Waghmare et al., 2019;Research, Society andDevelopment, v. 9, n. 10, e7579108896, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8896 as dessert, compote, frozen pulps, beverages, among others. The seeds can be baked or used in different recipes. Jackfruit seed powder is used as alternative flour in bakery and confectionary products by blending it with wheat flour and other low-cost flours. The lack of low cost and nutritive products led researches to seek for alternative protein sources which attend low cost production demands. Jackfruit seeds could be used as an economic alternative protein source to tackle the malnutrition (Waghmare et al., 2019).
In literature there is a lack of data on studies concerning protein properties of jackfruit seeds for technological applications, an obstacle for its utilization as a protein source on food production. Characterization of protein properties is necessary for underlying basic and applied studies about seed proteins. Such studies aim material suitability and applicability in new products in food industries and even as a protein supplement in food systems (Furtado et al., 2001).
According to Ajibola et al. (2016), solubility is an important requeriment for proteins to beuseful as functional ingredients in food. Solubility is affected by environmental conditions which influence its physico-chemical characteristics, such as pH, ions nature, concentration and strength, temperature, and the presence of organic solvents (Zayas, 1997), which reduce the medium dielectric constant and protein molecules hydration, causing denaturation and, consequently, reducing protein solubility (Ribeiro & Seravalli, 2007).
Concerning the importance of new products development, the search for alternative protein sources for food and beverage application, and the scarce utilization of jackfruit seeds, the objectives of this study are: to determine protein solubility of jackfruit (Artocarpus integrifolia L) seed flour; to evaluate the effect of pH and sulfate, chloride and trisodium citrate salts concentration on protein solubility.

Methodology
The present work was conducted in the Laboratory of Process Engineering at the Southwest Bahia State University (UESB). Jackfruits were obtained at the local market, washed, pealed, and pulp was removed. Seeds were washed in water, grinded in an industrial blender, sieved using a nº 10 sieve, and washed in running water. After filtration the residual part was oven dried in 20 o C for 24 h and grinded in order to obtain typical flour texture. In sequence, the product was stored in plastic containers under room temperature. Research, Society and Development, v. 9, n. 10, e7579108896, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8896 5

Effect of pH and salt concentration in jackfruit seed flour solubility
Solubility of jackfruit seed flour was determined using the methodology proposed by Machado et al. (2007), modifying pH values. 0.5 g of jackfruit seed flour of known protein content was weighed and 100 ml of buffer solution was added. Buffer solution of pH 4.0, 6.0 and 8.0 were adjusted with disodium phosphate (0.05M) and citric acid (0.05 M), hydrochloric acid (0.05 mol/L) and disodium phosphate (0.05M) in pH 2.0 and with disodium phosphate (0.05 mol/L) and sodium carbonate (0.05 mol/L) in pH 10.0. The material was magnetically stirred for 30 minutes at constant speed and room temperature (25 ± 3 °C). In sequence, the material was centrifuged at 1200xG for 30 min. Supernatant aliquots were collected for protein analysis according to the method used by Bradford (1976).
Salts of sodium chloride, sodium sulfate and trisodium citrate were used in concentrations of 0.25mol/L, 0.50 mol/L, 0.75 mol/L and 1.0 mol/L. Solubility (S, g protein/ 100 g solution) was defined as the protein amount in the supernatant, according to Equation 1: x 100 (1) Where: MP is the protein in the supernatant (g) and MS is the supernatant (g), calculated from the density found using the pycnometer method.

Experimental Design and Statistical Analysis
The experiment was conducted according to a completely random experimental design in a factorial block of five pH values, five salt concentrations and two repetitions.
Experimental data was analyzed using the procedure PROC GLM from SAS software (SAS version 9.1). Some were tested by Analysis of Variance, and once appropriate, through Turkey test for average comparisons. The level of significance was equal or lower than 5%.

Results and Discussion
Solubility was related to pH (2.0, 4.0, 6.0, 8.0, 10.0) and trisodium citrate, sodium chloride and sodium sulfate concentrations (0.25 mol/L, 0.50 mol/L, 0.75 mol/L and 1.0 mol/L). Research, Society and Development, v. 9, n. 10, e7579108896, 2020 (CC BY 4 Studies concerning characterization of additives and salts effect on protein solubility have been often conducted in order to understand solubility profiles for its application in food industries. In the present study, the influence of three salts on jackfruit seed flour solubility was analyzed: trisodium citrate, sodium chloride and sodium sulfate.

Trisodium Citrate
The effect of pH and trisodium citrate concentration on protein solubility of jackfruit seed flour is presented in Figure 1 In Figure 1 it is possible to observe that protein solubility slightly increases as salt concentration decreases, phenomenon which is not observed for most leguminous seeds, especially around the isoelectric region in which solubility is higher in the presence of salts, such as lentils (Lens Culinaris Medik) (Neves et al., 1998).
Solubility peak occurs in pH 6.0 and 1.0 mol/L and the lowest solubility is found in pH 8.0 and 0.25 mol/L, which promotes pI displacement. The behavior can be explained by the interaction between added salts and other substances used in pH correction solution. Research, Society and Development, v. 9, n. 10, e7579108896, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8896

Sodium Chloride
The effect of pH and sodium chloride concentration on protein solubility of jackfruit seed flour is presented in Figure 2.  Research, Society and Development, v. 9, n. 10, e7579108896, 2020 (CC BY 4 Solubility decreased with salt concentration increase in the system. Consequently, NaCl, which is the salt used more often for increasing protein solubility in most leguminous seed, did not present satisfactory results. Chickpea flour (Cicer arietnum L.) solubility was higher in the presence of 1.0 M NaCl (Silva et al., 2001).

Sodium Sulfate
The effect of pH and sodium sulfate concentration on protein solubility of jackfruit seed flour is shown in Figure 3. Within the concentrations under analysis there was a solubility increase in the isoelectric point, except for 1.0 mol/L, differing from the effects of trisodium citrate. This effect is observed for most of leguminous seeds, especially in the isoelectric region where solubility is higher in salt presence, such as sweet and sour lupine flour (El-Adawy et al., 2001), soy flour (McWatters & Holmes, 1979), sesame protein concentrates (Inyang & Iduh, 1996) and wheat gluten (Mejri et al., 2005).
It was also observed that the lowest solubility occurred in extreme pH values (basic or acid) under all concentrations. Sousa (2007) explained that this can occur because in extreme pH values protein can denature and expose more hydrophobic groups due to weakening of electrostatic bonds' forces. Therefore, protein molecules aggregate and precipitate.  Fontanari (2007) reported a decrease in solubility of guava seed protein isolate as sodium chloride concentration increased. According to León (2008), trisodium citrate Research, Society and Development, v. 9, n. 10, e7579108896, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8896 10 presented higher salting-out effect than sodium sulfate. The author stated that citrate ions decrease protein solubility due to an increase of hydrophobic interactions and molecule aggregation caused by ions competition for the water molecule. In this study the opposite occurred: the salt which caused higher solubility was trisodium citrate.
Some salts concentration decreased protein solubility of jackfruit seed flour. The fact can be explained by the salting out effect due to high concentration of some salts in protein solutions or due to the presence of other components in flour, such as carbohydrates and water soluble fibers. The models proposed by the Equations were significative (P<0.05) for explaining the emulsifying capacity variation as a function of pH and salt concentration, presenting R 2 =0.82 for sulfate and citrate and R 2 =0.99 for chloride.
Coefficients estimations for Equations 1, 2 and 3 are presented in Table 1.