Greek yogurt with added sorghum flours : antioxidant potential and sensory acceptance Yogurt griego adicionado

Este trabajo fue recibido el 10 de septiembre de 2019. Aceptado con modificaciones: 17 de octubre de 2019. Aceptado para ser publicado: 19 de noviembre de 2019. ABSTRACT The aim of this study was to develop and evaluate the antioxidant characteristics and sensory acceptance of Greek yogurt with added whole sorghum flours (WSF) from genotypes BRS305 (tannin) and BR501 (tannin-free). Five formulations were elaborated: 1) Control (without WSF); 2) 2% BR501; 3) 4% BR501; 4) 2% BRS305 and 5) 4% BRS305 and evaluated for antioxidant capacity, total anthocyanins, total phenols, condensed tannins, sensory acceptance and purchase intention. The 4% BRS305 formulation presented higher antioxidant properties, but lower sensory acceptance. No significant difference was observed for: color, flavor and overall acceptability between the control and 2% BR501; nor for texture and overall acceptability between the control and 4% BR501. Principal component analysis explained that all variables associated with antioxidant properties were positively correlated with the first major component (PC1: 82.7%). The 2% and 4% BR501 formulations were more highly correlated with PC1 (92.7%), as well as the control in relation to sensorial characteristics. The addition of WSF BRS305 improved the antioxidant properties of yogurts and the addition of WSF BR501 did not interfere with the sensory acceptance of the formulations. The similarity of the sensory acceptance of the yogurt containing WSF from the BR 501 genotype to the control, opens perspectives for the insertion of sorghum in human food, using dairy products as suitable matrices, adding potential functionality to this type of product. Keyword: Anthocyanins; Dairy products; Functional foods; Sorghum bicolor L. Moench; Tannins.


INTRODUCTION
The fermented yogurt food matrix provides increased absorption and nutrient digestion and its consumption is associated with reduced risk of developing cardiovascular diseases and obesity 1 . Greek yogurt (GY), also known as Labneh, originated in the Balkans 2 and is usually fermented by the symbiotic lactic acid culture of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, as well as traditional yogurt. GY is considered an intermediate product between fermented milk and fresh cheeses as the desorption of the curds after fermentation results in a concentrated product 2 .
On average, GY contains between 20.5-24.6% total solids, 6.4-10.7% total fat, 8.2-10.4% total protein, 1.1-1.3% minerals and a pH range of 3.67-4.05, having on average: 2 to 3 times more fat and protein content, 50% more minerals and a higher number of viable microorganisms compared to traditional yogurts. GY has excellent creaminess, with little syneresis, is slight acidity around 1.80-2.00% and low lactose concentration (about 6.00%) 2 . The health benefits of yogurt may be even greater when paired with fruit or cereals rich in fiber and bioactive compounds 3 .
The addition of fiber in yogurts can help to maintain the viability of probiotics in the product 4 by providing a substrate and conferring health benefits to hosts with constipation problems 5 . It is also known that whole grains present nutritional superiority due to the presence of embryo and bran grain structures which, besides fiber, contain: several minerals, vitamins and bioactive compounds. The consumption of whole grains is therefore recommended by nutritionists and other health professionals over the consumption of refined grains 6,7 .
In this context, the incorporation of whole sorghum flour (WSF) (Sorghum bicolor L.) into GY may potentially result in a viable product alternative with functional properties.
Sorghum is a cereal that has significant concentrations of dietary fiber, minerals (iron, calcium, potassium) and vitamins (riboflavin, thiamine) 8,9 ; in addition to bioactive compounds such as: phytosterols, policosanols, resistant starch, anthocyanins and condensed tannins 9,10 . Some of these compounds stand out for having a high antioxidant capacity, acting against the deleterious effects of free radicals in the organism 11,12,13 . Moreover, sorghum is gluten-free 13,14 , making it a safe alternative for people with celiac disease and those with gluten intolerance 15,16,17 .
However, it is known that some genotypes of this cereal may present differences in their chemical and nutritional composition 8,18 , which can lead to technological and sensorial changes to the products to which they are added. Still, there are few studies that add sorghum to the milk matrix 16 ; and none that compare the addition of different genotypes.
Thus, the aim of this study was to develop GY with added WSF of genotypes BRS305 (with tannin) and BR501 (tannin-free) and to evaluate antioxidant characteristics and sensorial acceptance.

MATERIAL AND METHODS
The experiment was carried out in laboratories of the Food Engineering Department of the Universidade Federal de São João del-Rei, (Sete Lagoas, Minas Gerais, Brazil) and at Embrapa Milho e Sorgo (Sete Lagoas, Minas Gerais, Brazil).

Whole sorghum flours (WSF) preparation
Two sorghum genotypes were selected based on their differences in pericarp color, chemical composition and bioactive compounds 8,10,19 . The BR 501 genotype has a white pericarp and no pigmented testa (tannin-free) and BRS 305 genotype has a brown pericarp and pigmented testa (with tannin). The preparation of WSF was carried out at Embrapa Milho e Sorgo with the following steps: 1) Manual selection of grains; 2) Sieving to remove dirt and impurities; 3) Milling in Hawos Mill, IKA model A11 Basic, for granulometry 0.5 mm; 4) Packing in polyethylene plastic bags, protected from the light; 5) Storage under refrigeration (4 °C) until GY processing and chemical analysis.
The proximate composition were performed, in triplicate, on WSF, according to AOAC 20 and the lipid content followed the AOCS protocol 21 . Neutral Detergent Fiber (NDF) was analyzed in 0.500 g of sample with a Tecnal EQ LCC 08 fiber analyzer using the Ankom system with filter bags 22 .

Greek yogurt (GY) processing
The ingredients for GY processing (pasteurized whole milk, skimmed milk powder, crystal sugar and lactic yeast) were purchased at local market in Sete Lagoas, MG, Brazil. The lactic yeast (Docina Nutrição LTDA) contained the thermophilic cultures S. thermophilus and L. bulgaricus.
Five formulations of GY were developed: 1) Control (without addition of WSF); 2) 2% WSF from BR 501 genotype; 3) 4% WSF from BR 501 genotype; 4) 2% WSF from BRS 305 genotype and, 5) 4% WSF from BRS 305 genotype. The formulations were developed according to figure 1. The WSF concentrations were based on previous tests that subjectively evaluated the influence of different incorporation percentages on the yogurt texture. Texture is reported as one of the primary factors that determine the quality and acceptability of yogurt in the consumer market 23 . The AC was determined by the ABTS assay (2,2-azinobis-[3-etil-benzotiazoline-6-sulfonate] radical cation (ABTS)) according to the method presented by Awika et al. 24 . The results were expressed as μmol Trolox.g −1 , on a dry basis (db).
The method used for TA determination was that described by Fuleki & Francis 25 , and further detailed by Awika & Rooney 27 . The concentrations of 3-deoxiantocianinas were calculated based on the absorbance of luteolinidin (480 nm) using the eq. C (mol/L)= A/ε, where C is sample concentration, A is the absorbance and ε is the molar extinction coefficient of luteolinidin. The molar extinction coefficient of luteolinidin used was 29.157 (Njongmeta, 2009). The results were expressed as mg luteolinidin equivalentes (LE)/g sample, on dry basis (db).
The TP was determined according to the method proposed by Kaluza et al. 28 and further detailed by Dykes et al. 29 , using Folin Ciocalteau reagent. The results were calculated and expressed as mg gallic acid equivalent (GAE)/g sample, on a dry basis (db).
For CT determination, the Vanillin/HCl reaction method described by Price et al. 30 was used. The results were calculated and expressed as mg catechin equivalent (CE)/g sample, on a dry basis (db).

Sensory acceptance
The sensory tests to evaluate the acceptance of the GY with added tannin (BRS 305) and tannin-free (BR 501) WSF were carried out in the Sensory Analysis Laboratory of the Federal University of São João del Rei, (Sete Lagoas, Minas Gerais, Brazil). A total of 100 non-trained consumers, between 18 and 63 years of age, evaluated the sensory acceptability of the products.
The The GY samples were served in 50 mL white plastic cups coded with three-digit numbers, in complete blocks, in a monadic, sequential and random order, in a single test session. Each consumer evaluated the attributes for: color, aroma, flavor, texture, and overall acceptability, using a 9-point hedonic scale (9= extremely like, 1= extremely dislike). The purchase intention was also evaluated using a 5-point scale (5= certainly would buy the product, 1= certainly would not buy the product).
Additionally, the acceptance index (AI) was calculated for each sensory attribute according to the formula 32 .
Where, AI= acceptance index A= acceptance test results average B= highest score obtained in the acceptance test The AI was classified into three groups 16 : (1) rejection <50.0%; (2) indifference between 50 and 59.0%; (3) acceptance ≥ 60.0%. The results were expressed in relative frequency.

Statistical Analyses
Data were analyzed using the software R version 3.5.3. The results were compared by analysis of variance (ANOVA), considering p<0.05 as significant. The Tukey test was used to determine significant differences among formulations (p< 0.05). Results were expressed as mean ± standard deviation. The AI data were analyzed by analysis of the relative frequency distribution.
The principal component analysis (PCA) was performed to evaluate the correlations of the addition of sorghum genotypes to the antioxidant and sensory variables of the formulations. Strong correlations were considered between 0.700-1.00 33 .

RESULTS
Antioxidant properties: total anthocyanins (TA), total phenols (TP), condensed tannins (CT) and antioxidant capacity (AC) Table 1 shows the AC and the TA, TP and CT concentrations for the GY formulations developed in this study.
The 4% BRS 305 formulation presented higher AC and higher TA, TP, CT contents when compared to the others (p< 0.05).
The TA content differed among all formulations (p<0.05, Table 1), with higher scores for those with added WSF from the BRS 305 genotype. Only the 2% BR 501 formulation did not differ from the control (p> 0.05) in relation to TA concentration, both presenting the lowest values when compared to the others.

Sensory acceptance
The sensory acceptance and purchase intention for the GY formulations are shown in table 2.
The control formulation obtained the highest mean scores for all sensory attributes and for purchase intent. However, there was no difference observed among the control formulation and the 2% BR 501 and 4% BR 501 formulations for the color and overall acceptability attributes. The control formulation also did not differ from the 2% BR 501 for flavor and 4% BR 501 for texture (p> 0.05, Table 2).
The mean scores for flavor of all the formulations are situated between the "I liked it" and "indifferent" hedonic terms, with AI, varying between 80.2% (control) and 60.3% (4% BRS 305).
On the other hand, the 4% BRS 305 formulation differed from the others in all sensory attributes evaluated (p<0.05), except for aroma (p> 0.05), presenting the lowest acceptance mean scores. The 2% BRS 305 formulation differed from the control for color and texture (p<0.05).
For the texture attribute, the control formulation and the 4% BR 501 formulation were the most accepted, with AI of 84.1% and 78.2%, respectively. On the other hand, the formulations with the WSF from the BRS 305 genotype obtained the lowest AI, 74.6% (2% BRS 305) and 63.1% (4% BRS 305).
Regarding overall acceptability, average scores were between the terms "I liked it very much" and "I liked it slightly", with AI varying between 83.8% (control) and 68.7% (4% BRS 305). The yogurts with added WSF from the BR 501 genotype were the most accepted, with AI of 79.8% (2% BR 501) and 77.9% (4% BR 501). The 4% BRS 305 formulation was the least accepted (AI= 68.7%). The formulations with lower concentrations of either WSF genotypes did not differ in relation to the overall acceptability attribute when compared to the control (p> 0.05).
Considering the purchase intention, only the 4% BRS 305 formulation was included in the "would not buy" range, differing from the others that were between the intentions "possibly would buy" and "maybe would buy." This result confirms their lower acceptance ( Table 2). The 2% BRS 305 formulation did not differ in the intention to buy rankings when compared to the formulations with the WSF from the BR 501 genotype (p> 0.05), as their results were ranked between the terms "possibly buy" and "maybe buy". Figure 2 shows the PCA for the antioxidant characteristics and sensorial attributes of Greek yogurt.

Principal Component Analysis (PCA)
Two principal components (PC1 and PC2) were used and together they explained 91.3% of the total variance of the antioxidant characteristics of the formulations; PC1 explained most of the variations (82.7%), all variables being positively correlated with PC1 ( Figure 2B). The 4% BRS 305 formulation was characterized by a higher antioxidant content, while the other formulations presented similar antioxidant characteristics (Figure 2A).
Regarding sensory acceptance, the PCA showed that a principal component (PC1) explained 92.7% of the data

DISCUSSION
The BRS 305 sorghum genotype has higher CT concentrations naturally 19 . Dykes et al. 29 and Wu et al. 34 reported that tannins have been identified as the main compounds that contribute to the increase of AC and TP in some sorghum genotypes. This explains the higher antioxidant properties presented in the GY formulations developed with sorghum genotypes in this study.
This genotype has brown pericarp and pigmented testa, which can result in alterations of color and flavor in the products to which it is added. Tannins are known to present an astringent and bitter residual flavor, which may compromise the palatability and product acceptance 27,35 . Therefore, the lower sensorial acceptance presented by the 4% BRS 305 formulation can be explained by the higher CT concentration found in this formulation (Table 1).
Queiroz et al. 16 developed a powder mixture for the preparation of beverages added with 56.7% of extruded sorghum flour, where the formulation containing tannin sorghum of the PDT-3670 genotype was more sensorially accepted for the attributes of flavor and overall acceptability than that containing tannin-free sorghum flour of the PDTF-7064 genotype.
These authors suggest that not all sorghum tannins negatively affect consumer acceptance and that other factors may also influence the acceptance of sorghum-added products such as grain processing (raw or extruded flour) and other ingredients which are present in the formulation of the product. The presence of polysaccharides, such as xanthan gums, for example, can react with tannins and reduce the sensation of astringency, leaving the product more palatable 16 . In addition, depending on the type of preparation, the condensed tannins may influence, to a greater or lesser degree, the sensory aspects of the product. In the present work, GY was formulated with non-extruded whole meal and may have undergone a greater negative influence of the addition of sorghum flour with tannins (BRS 305) than the beverage produced with extruded sorghum flour elaborated by Queiroz et al. 16 .
Additionally, Queiroz et al. 16 verified that the AC, TA, TP and CT concentrations were higher in a powdered beverage made with 56.7% of extruded sorghum flour containing tannin from the PDT-3670 genotype. This suggests a greater functional potential compared to the added formulation of sorghum without tannin.
It stands out that, although the indication that the presence of tannins in foods reduces protein digestibility and food efficiency in humans and animals 36 , the benefits of eating diets rich in tannins has highlighted because of an association with immunomodulatory, anticancer, antioxidant, anti-inflammatory, cardioprotective, vasodilator, and antithrombotic effects 11,36,37 . Thus, various research and a niche markets have emerged to capitalize on the benefits of sorghum with tannins through the development of products with higher concentrations of antioxidants and bioactive compounds 15,16,17,38,39,40,41 .
It is known that anthocyanins, besides being bioactive compounds with antioxidant, cardioprotective, anticancer, anti-obesity, anti-inflammatory, anti-diabetic and antimicrobial properties, are red, purple and blue pigments that can be used as natural food colorants, in the form of extract, or even by adding their food sources 42 .
Hernandez-Rodas et al. 43 reported the promising effect of some polyphenols, including anthocyanins in the prevention and treatment of nonalcoholic hepatic steatosis and its complications. These authors have described some mechanisms for reducing hepatocellular lipid accumulation and its deleterious effects on the organism: 1) inhibition of lipogenesis by decreasing SREBP-1c; 2) stimulation of lipolysis by inducing activity of PPAR-α and 3) reduction of oxidative stress. In association with anthocyanins and other phenolic compounds, the inclusion of sorghum in dairy matrices may enhance the benefits to human health by the presence of other natural antioxidants such as vitamin E, zinc and other compounds such as fiber and resistant starch.
Considering that the formulation 4% BR 501 presented higher concentrations of TA than the control formulation (p<0.05, Table 1), it is therefore suggested that the development of dairy products with added WSF constitutes a differentiated option for insertion into the market as these products are nutritionally richer and well accepted, regardless of the genotype of sorghum used.
For the texture attribute, the control formulation and the 4% BR 501 formulation were the most accepted. These formulations showed greater firmness, which probably affected the consumers perception for texture and palatability. Texture is reported as one of the primary factors that determine the quality and acceptability of this type of product in the consumer market 23 . The BR 501 genotype endosperm is more vitreous with firmer grain texture, resulting in a thicker flour, which may have influenced the development of a firmer textured product; whereas that the genotype BRS 305 presents smaller and softer grains, with more farinaceous endosperm, resulting in products with a softer texture 8,19,41,44 .
Curti et al. 45 and Hasani et al. 4 added, respectively, quinoa flour at concentrations of 1.0%, 3.0% and 5.0% and barley bran at concentrations of 0.300%, 0.600%, 0.900% and 1.20% in yogurts and verified that higher cereal concentrations can affect texture as well as influence flavor and aroma, and thus contribute to product rejection. In general, the addition of cereals in yogurts causes a sandy texture, influenced by the type of cereal and its concentration 3,46 . However, in the present work, there was a greater influence of the sorghum genotype rather than that of the added concentrations.
In relation to overall acceptability, the results suggest that the addition of 2.0% of sorghum flour, independent of the genotype, did not significantly alter the majority of the sensorial characteristics of the GY, therefore this concentration of WSF may be indicated for continued studies involving the addition of sorghum in dairy products. Although the 4% BRS 305 formulation presented the lowest overall acceptability, the AI (68.7%) of this formulation was compatible with a percentage of acceptance proposed by Queiroz et al. 16 where preparations with AI≥ 60.0% are considered acceptable.
According to Curti et al. 45 , overall acceptability is a complex term that comprises the combination of different sensory perceptions. In this context, the rejection by the consumer of any of the sensorial characteristics can negatively influence the general perception of the yogurt.
According to Prado et al. 41 the use of PCA provides a basis for determining patterns or data structure, as well as identifying the importance of individual variables in studies, thus facilitating the understanding of the dataset.
The results of the PCA in this study can be explained by the higher amount of WSF from the BRS 305 genotype added to the 4% BRS 305 formulation, associated with the antioxidant characteristics of this WSF with a higher CT and TA content ( Table 1).
The 2% BR 501 formulation has the most similar characteristics to those of the control, confirmed by the results shown by the PCA (Figure 2) and therefore, from the point of view of the sensorial acceptance, would be the most adequate for industrial production.
Therefore, when evaluating GY formulations in a holistic manner, the addition of 4% WSF from the BRS 305 genotype was superior to the other formulations in relation to the antioxidant characteristics. However, in relation to the sensorial attributes, this WSF genotype negatively influenced the scores given by consumers, as well as their intention to purchase this product; while the GY formulations with the addition of WSF from the BR 501 genotype were closer to the sensory characteristics of the control formulation. Still the 4% BR 501 formulation presented higher content of anthocyanins than the control formulation, which indicates perspectives for the development of dairy products added with WSF with potential functionality.
According to Fernandez et al. 1 , the chemical composition of yogurt associated with its fermented matrix can provide benefits with respect to the control of cardiometabolic diseases. The matrix effect of a food goes beyond individual nutrients, suggesting that the physical structure created by a combination of nutritional components, can act independently of its individual components during digestion and metabolism 1 . Furthermore, Lamothe et al. 47 reported that the dairy matrix structure can assist in maintaining and integrity of the antioxidant activity of polyphenols present in beverages such as green tea during digestion.
Additionally, the incorporation of fiber-rich whole grains may have additional effects on yogurt functionality 6 ; thus, the addition of sorghum in yogurt represents the possibility of developing differentiated products. These products have more fiber, mainly insoluble fiber, more bioactive compounds such as condensed tannins and anthocyanins, vitamin E and various minerals such as iron, phosphorus, magnesium and zinc 8 . Thus, in addition to the benefits usually associated with yogurt, it can contribute to a healthier diet, allowing improved traffic and intestinal health and preventing cardiovascular disease, cancer, obesity, high blood pressure, diabetes mellitus, hepatic steatosis, among others.

CONCLUSION
This work represents the first scientific approach of the incorporation of whole sorghum flour from different genotypes into Greek yogurt on the antioxidant and sensorial properties. The addition of these whole flours, especially the BRS 305 genotype, improved the antioxidant properties of the formulations developed. However, specific characteristics of this genotype, such as a higher concentration of condensed tannins, probably negatively influenced the sensory acceptance and purchase intention of these formulations. On the other hand, the similarity of the sensory acceptance of the yogurt containing the WSF from the BR 501 genotype to the control, opens perspectives for the insertion of sorghum in human food, using dairy products as suitable matrices, adding potential functionality to this type of product. Therefore, more studies are suggested to better investigate the technological advantages of the incorporation of sorghum into fermented dairy products, adjusting flour concentrations to balance the nutritional quality and the sensorial aspects of the products.