Development of yogurts with mixture of sheep and bovine milk: effect on chemical physical characteristics, protein profile and antioxidant activity

Yogurt is one of the most popular fermented dairy products and an excellent medium for new ingredients/food source. This study sought to evaluate and characterize bovine milk (BM) and sheep milk (SM) and yogurt made from bovine (BM) and sheep (SM) milk, and BM:SM mixture with the respective 100:0 fractions: 75:25, 50:50, 25:75 and 0:100, and evaluate their influence on physicochemical parameters, antioxidant, protein profile, microstructure and color analysis for yogurts. Yogurts with higher levels of acidity, protein, fat, total solids, ash and calcium were the ones made from SM.. Regarding the color attributes, all yogurts showed yellow-green characteristics after 21 days of storage by evaluating parameters a* and b*, with a predominance of bovine milk. The antioxidant profile showed a higher concentration at 100:0 and 0:100, but bovine milk has a higher DPPH (2,2-diphenyl-1-picrylhydrazyl) value, while yogurts maintained constant DPPH values during storage, being higher at 0:100. Scanning electron microscopy (SEM) showed that BM (100:0) has a more compact structure, possibly due to the larger size of the fat particles and the lower protein concentration compared to SM (0:100). Sheep milk presented better nutritional value, showing its potential in the manufacture of products.


Introduction
Yogurt is a fermented product produced from milk by the action of lactic acid bacteria as Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus et al. (Karnopp et al., 2017;Jørgensen et al., 2019;Fazilah et al., 2018;Costa et al., 2015;Costa et al., 2017). This product presents high protein content and convenience of consumption (Mohammadi-Gouraji, Soleimanian-Sad, & Ghiaci, 2019), with exponential growth in the world when compared to other dairy products, as well as the research development (Aryana & Olson, 2017). This can be attributed to health benefits, considering their regular intake (Mior, Novello, & Dinon, 2016). Thus, the use of differentiated raw materials, such as sheep milk (SM), may result in particular characteristics to yogurt, in relation to the source of high-quality proteins, calcium and lipids (Abreu et al., 2018;Tribst et al., 2018) and adding value to the product, which comes in line with the current scenario of the consumer market.
Sheep milk products highlight the nutritional and organoleptic traits due to the higher protein and solids content, linolenic acid, conjugated linoleic acid (CLA) (between 0.405 and 1,250 g CLA/100 g fat), essential amino acids, vitamins and minerals, easy digestibility and hypo allergenicity, immunoregulatory effect and activity such as antiobesity, antioxidant and antidiabetic when compared to bovine dairy products (Feng et al., 2019;Kaminarides, Stamou, & Massouras, 2007;Yuan, Chen, & Li, 2014;Wang & Lee, 2015;Tribst et al., 2020a). Yogurt and other dairy products using SM have higher creaminess and stability compared to bovine milk (BM) (Tribst et al., 2018). Due to these properties, the interest is growing in SM yogurt, considering its nutritional value and sensory characteristics (Zamberlin & Samaržija, 2017;Tribst et al., 2020a).
However, the amount of sheep dairy products is limited due to low and seasonal milk production throughout the year and a short lactation period (Tribst, Falcade, & Oliveira, 2019;Tribst et al., 2020b). Studies with use of milk of different species, as well as mixtures of milk types with nearby physical-chemical characteristics may result in products with interesting characteristics such as protein, mineral and fat content, since it has easy digestion, containing small fat globules that provide greater contact area for digestive enzymes, making their use more efficient compared to other milks (Revers et al., 2016;Roy et al., 2020). These different flavor and nutritional profiles can stimulate the consumption of these yogurts (Rettedal et al., 2019;Abreu et al., 2016).
Regarding the BM, it was obtained from crossbreed Holstein and Jersey (Jersolando), reared in pasture system with supplementation of corn silage concentrate, soybean meal and vitamins and minerals complex. The Lacaune ewes were reared in confinement system, with balanced feeding composed of hay and pre-dried grass, corn silage concentrate, soybean meal and vitamin and mineral complex.
All milks and their proportions were pasteurized and analyzed for pH (method 943.02) (mPA 210 -MS Tecnopon), acidity (method 947.05), lactose by Lane -Eynon (by redox titration using alkaline CuSO4), in agreement with the Association of Official Analytical Chemistry -AOAC (2016).
The phenolic content of the yogurt samples 100: 0, 75:25, 50:50, 25:75, 0:100 were determined according to the modified Folin-Ciocalteu method procedure (Wang et al., 2011). Data were expressed as equivalents of gallic acid in mg per 100 g of weight (mg GAE.100g-1). Both activities were determined in the yogurts on the 1st and 21st days of storage.

Statistical analysis
The results obtained from the analyses were statistically evaluated by the Tukey Test, through variance analysis (ANOVA), using statistic 10.0 software to detect the significant difference between samples, at a 95% confidence level.

Physicochemical analyses of milk
The results of the physicochemical analyses of milk and their mixtures (Table 1)   In relation to pH, BM presented values higher than SM (p<0.05) and consequently lower acidity (p<0.05). The same behavior can be observed in mixtures where the proportion of BM in SM was lower (Table 1). The pH and acidity values of sheep milk can be influenced by production in different seasons, with pH ~ 6,58 at the winter (Malta et al., 2021), value similar to 0:100 (BM:SM). But the 100:0 was the even to 75:25 and 50:50 (p<0.05). Similar behavior was observed for milk mixtures BM:SM (100:0) pH 6,77 and 6,41 (0:100) (Lima et al., 2020).
The lactose content showed statistical difference (p<0.05), from the mixtures of major proportion of sheep milk (25:75 and 0:100). Lactose content is one of the compounds that has lower sensitivity for variation due to seasonality. However Fava et al. (2014) observed quantitative changes in SM lactose throughout the year ranging from 4.17 ± 0.4 to 4.60 ± 0.16 %. Lactose also varies throughout the lactation stage for all ruminants, presenting lower levels at the beginning and end of lactation (Park et al., 2007). Lactose showed no significant difference (P < 0.05) between the seasons of the year ~ 5 % (Malta et al., 2021) and similar tendency was observed for milk mixtures BM:SM (100:0) 3,74 and 4,26 (0:100) (Lima et al., 2020). The values found in this study were close to those obtained by Nguyen et al. (2013) to lactose content, 4.40%, similar to 0:100 (BM:SM).

Fermentation time
The fermentation time of yogurt was affected in relation to the type of milk, with the longest time of 330 min for SM compared to 240 min for BM ( Figure 1), following the same behavior for yogurt with milk mixtures, and the highest concentrations of SM required longer periods to reach pH 4.7. The yogurt that had the highest concentration of SM (0:100) presented the highest fermentation time (330 min) and the highest titratable acidity value (0.79 % lactic acid), being the lowest value of acidity obtained for yogurt with BM (100:0) (0.59 % lactic acid / 265 min). These differences are possibly associated with higher levels of total solids, especially sheep milk protein (Asensio-Vegas et al., 2018), which has a direct effect on milk structure and the mechanism of action of milk culture.
During yogurt fermentation, the living microorganisms of the initial culture added perform metabolic activities that modify the physical and nutritional characteristics, decomposing complex components and forming subproducts (Rettedal et al., 2019), acting mainly on the lactose present in milk, breaking its structures and producing organic acid molecules, such as lactic acid (Pereira da Sultan et al., 2016), stage in which texture and flavor characteristics are developed (Bett et al., 2017). Table 2 presents the results of physical-chemical composition of yogurts produced from SM and BM and their mixtures. Yogurts differed statistically for the variables protein, fat, total solids, ash and calcium (p<0.05), increasing values as the proportion of sheep milk increased (Table 2).

Physicochemical analyses of yogurts
A study conducted by Vianna et al. (2017), showed this same trend, i.e. bovine milk yogurts had lower protein content (3.39 ± 0.00 %) when compared to sheep yogurt (5.25 ± 0.00 %). The higher protein content of sheep yogurt in relation to bovine yogurt is due to the compositions of both milks, being characteristic with higher protein contents in sheep milk (Pandya & Ghodke, 2007;De La Vara et al., 2018). Serra et al. (2009), followed the shelf life of bovine milk yogurts, noting the hydrolysis of casein and the increase of soluble nitrogen, which consequently means a decrease in the total protein content after 28 days of storage.
The lipid compositions of yogurt mixtures showed statistical difference (p<0.05), demonstrating an increase in the fat content caused by the addition of sheep milk ( Table 2). The characteristics of sheep milk fat globules have a smaller diameter, around 3.5 μm, while bovine milk has a diameter of 4 μm . In the manufacture of yogurt, the small uniform fat globules of sheep milk ensure in the processing homogenization and dispersion, avoiding the separation of the cream into yogurt (Kalyankar et al., 2016). The diameter of fat globules also influences digestive capacity, i.e., in milks with a larger globular diameter, lipid metabolism acts less efficiently (Gantner et al., 2015), being slowly digestible.
The total solids parameter showed statistical difference (p<0.05), with an increase in the values in the mixtures along the addition of sheep milk, directly related to the higher protein and fat content (  Eissa, Babiker and Yagoub (2011). The difference between the chemical compositions between milks is related to several factors, as described by Park et al. (2007), such as lactation period, age and breed of animals, milking intervals, season, temperature, nutrients from the diet and diseases in the udder.
The mineral content available in yogurt is directly related to the content contained mineral in the raw material. In this study, only the analysis of calcium content was for its predominance, where according to studies (Park et al., 2007;Payandeh et al., 2015), the mineral calcium content in bovine milk can range from 107 to 133 mg.100 mL-1 and 136 to 218.44 mg.100 mL-1 for sheep milk.
The highest calcium contents were found in yogurt samples (25:75) with 573.06 + 11.02 mg.L -1 and yogurt (0:100)  The content total solids, ash, minerals and other nutrients the sheep's milk may undergo greater variations when compared to bovine milk. These changes are influenced by lactation stage, animal nutritional status, environmental and genetic factors caused by feeding and seasonal variations (Kalyankar et al., 2016).

Titratable acidity and pH of yogurts
Titratable acidity increased gradually (Figure 2A) while pH values showed a slight decreasing trend ( Figure 2B) for all yogurts during the storage period of 21 days at 4 ± 1 ° C. This behavior is related to milk cultures added to yogurt, which can develop on a smaller scale in adverse conditions such as storage temperature, producing organic acid and altering chemical characteristics of the product, such as pH and acidity.
Yogurt 100:0 prepared only by BM resulted in an increase in significant acidity (p<0.05) between the 1st day and the 21st day of storage from 0.59 ± 0.01 % lactic acid to 0.75 ± 0.01% lactic acid, respectively. Sheep yogurt (0:100) showed differences in more expressive acidity values (p<0.05), 0.98 ± 0.07% lactic acid and 1.21 ± 0.05% lactic acid for 1st day and 21st days of storage, respectively. Significant differences in acidity between formulations during the storage period (p<0.05) are observed, since milk from different species has different compositions of constituents that have effects on the process (Rettedal et al., 2019). As in the raw material, the milk mixtures with the presence of sheep milk in yogurts (75:25, 50:50 and 25:75) present higher acidity when compared to yogurt (100:0) in different storage times. Similar results are observed in the study by Sultan et al., (2016) to obtain yogurts with milk of different species (cow, sheep, goat and buffalo) for 10 days of storage. The pH change after fermentation processing (Figure 2A), where the standard pH was 4.7 can be evidenced by its difference compared to the first day of storage ( Figure 2B). It is observed that the pH values did not have great variations between analyzed times (p<0.05) between the samples, but it is worth mentioning that the pH of yogurt can influence the syneresis process, which increases when the values are below 4.7, resulting from ferroprotein that are soluble, and milk caseins that do not remain at the isoelectric point (PI) (Farrell et al., 2006). After 24 hours of the preparation of yogurts, the lowest value of *L, related to the yogurt sample (0:100) ( Table 3),

Color Analysis
suggests that the greater amount of total solids found for this sample (Table 2) may favor the absorption and reduction of free water, with a lower reflection of light (García-Pérez et al., 2005).
L* values elevated after 21 days of storage are correlated with yogurt whiteness that is associated with yogurt refrigeration after fermentation (Peixoto et al., 2016;García-Pérez et al., 2005).
The values of a* did not differ significantly (p>0.05) between the samples during storage time and showed negative values which the yogurt is shown as greenish (Table 3). Research, Society andDevelopment, v. 11, n. 16, e192111637531, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i16.37531 9 For parameter b* there was a significant difference between the evaluated samples (p<0.05), (Table 3). The yogurt prepared with only bovine milk (100:0) presented the highest value of b*, statistically equivalent to the values obtained for the mixtures and consequently greater tendency to yellowing. Sheep milk has higher amounts of vitamin A than bovine milk (Park et al., 2007). Sheep and goats convert all β-carotene into retinol in milk, resulting in whiter milks (Mohammadi-Gouraji et al., 2019) opposing the more yellowish color caused by the carotenoids of bovine milk .

Antioxidant activity
Studies have shown that casein and whey proteins have antioxidant properties, presumably based on their ability to bind transition metals and eliminate free radicals (Tong et al., 2000).  The different behaviors of antioxidant compounds are defined according to the environment in which they are found (Cottica et al., 2018), either in pure milk or in mixtures. According to Rashidi et al. (2016), antioxidants mostly work synergistically in the presence of other antioxidants.
Many factors, such as antioxidant concentration, temperature, pH and processing treatment, can influence the antioxidant activity of the products (Gazzani et al., 1998). The yogurts 50:50 and 25:75 did not differ significantly (p<0.05) for the DPPH values, since the concentrations of sheep and bovine milk resulted in less variation of the antioxidant factor during storage.
The DPPH radical elimination activity expresses the potential of yogurt substances to act as electron or hydrogen donors in the stable DPPH radical. According to studies by Brand-Williams, Cuvelier and Berset (1995) According to Farvin et al. (2010) the oxidative stability of yogurt may be due to antioxidant peptides released during milk fermentation by lactic acid bacteria, we can consider the formation of new substances, with antioxidant activity. During storage of yogurts Lima et al., (2014) also found an increase in antioxidant activity. This behavior may explain the similarity and stability in the percentage of radical elimination activity for the yogurt samples after storage for 21 days. Furthermore, nutrients can have their total amount changed due to chemical degradation caused by storage and can promote the release of new compounds, causing effects on the availability of active compounds (Caleja et al., 2016).
Polyphenols are part of the antioxidant group. Ruminants have a mechanism of detoxification of phenolic compounds by microorganisms in the rumen (Gagnon et al., 2009), and this indicates that concentrations of polyphenols secreted in milk are very low. BM (100:0) exhibited the highest value in phenolics, which cooperated for the total percentage of radical elimination activity. All samples showed a significant difference (p<0.05) from each other.
Storage time significantly affects the maintenance of phenolic compounds in all yogurts. A decrease in phenolic content was observed in all yogurt samples tested. Similar results were found in studies by Karaaslan et al. (2011), where they tested the addition of four varieties of grapes in yogurt by analyzing contents of phenolic compounds in storage. Phenolic compounds are easily degradable and form high reactivity compounds, in which the reduction in phenolic content is related to the decline in the antioxidant capacity of yogurts. The samples did not differ significantly differences (p<0.05) in the 21 days of storage.

Scanning Electron Microscopy (SEM)
Sheep milk has smaller fat globules and also due to the higher protein content, it remains a less compact structure than bovine milk (Figure 4). Bovine milk with rounded forms, lower fat and protein content leave the structure with small imperfections and more compact, as shown by 100:0) and 75:25 that has higher bovine milk content in its composition. In all yogurt gels, protein nets were formed after acidification of the medium ( Figure 5). The microstructures of yogurts (100:0, 75:25, 50:50, 25:75 and 0:100), which consist of a three-dimensional network of chain and aggregates of casein micelles in which the 100:0 yogurt gel exhibited a more porous microstructure and less dense protein network, while the yogurt gel 0:100 has a more homogeneous structure (Nguyen, Afsar, & Day, 2018), with the most defined crevices, being observable in yogurts 25:75 and 0:100 with greater proportion of SM, with globular shape, interspersed with empty zones (Nguyen, Afsar & Day, 2018;Bramanti et al., 2003).
The structural difference displayed in yogurts is the result of the different physical-chemical properties of dairy materials used in yogurt production (Nguyen, Afsar & Day, 2018), considering what water retention capacity, is dependent increase dry matter, protein and fat contents (Gregersen et al., 2021), in addition in which acidification was induced by the fermentation of lactic acid bacteria, may be justification.
The similarity between 100:0 and 75:25 is remarkable, as well as between 25:75 and 0:100, demonstrating that the organization of the yogurt gel was influenced by the proportion of the type of milk used.

Electrophoresis
The behavior of proteins in BM, SM and their mixtures and also in yogurts with respective mixtures, by the relationship of electrophoretic profiles, obtained with the addition of SDS (sodium dodecyl sulfate, anionic detergent). The relationship between standard molecular weight (P) and protein bands are identified as immunoglobulins include the range of 150 -1,000 kDa, lactoperoxidase (75 kDa), α s-casein (35 kDa), β-casein (25 kDa), β-lactoglobulin (15 kDa), α-lactalbumin (10 kDa) ( Figure   6), fractions vary in molecular weight (PM), isoelectric point and phosphorylation level. The division between α and β-casein is hardly visible in electrophoresis gel due to the greater amount of these proteins in milk , the darker intensity band is considered by the largest fraction of caseins (α s-casein and β-casein) observed in whole milks and their respective mixtures. The possible differences in the fraction of the main casein proteins and molecular structures of the SM proteins ( Figure 5), result in the different structures of the yogurts, and there may be interference in the gelation and firmness of the yogurt.
The largest fraction of caseins of milk due to the transformations that occur during fermentation and yogurt production, were possibly transformed into peptides of molecular weight less than 10kD (Preci et al., 2021).
β-lactoglobulin is present in greater amounts in SM than other whey proteins (immunoglobulin, lactoferrin, αlactalbumin) (Selvaggi et al., 2014;Balthazar et al., 2018), which may have contributed to the stability of the gel and the lower syneresis observed especially for yogurt 0:100. The, camel milk no β-lactoglobulin, camel yoghurt has difficulty forming coagulum during fermentation compared with the cow yoghurt. Lack of β-lactoglobulin makes it more difficult to make camel milk yoghurt (Konuspayeva, 2020).
Other characteristics of SM proteins are smaller in size (193 nm) than BM (260 nm) (Park et al., 2007). Therefore, SM has lower allergenicity (Dias et al., 2022;Gantner et al., 2015). According to Masoodi and Shafi (2010), αS1 and s2 αS2 protein sequences of SM differ sharply from αS1 and protein sequence αS2 of BM. This fact suggests that SM promotes lower allergic sensitization. In addition, it has 99% similarity between the protein sequences of casein αS1 and αS2 of goat's milk, being less allergenic than bovine milk (Masoodi & Shafi, 2010).

Conclusion
The parameters analyzed demonstrated the individuality and distinct characteristics of each type of milk and mixtures.
It was observed that sheep milk presented values for physical-chemical characteristics, in almost all parameters higher than those of bovine milk, and the same behavior was observed for yogurts. It was verified that the antioxidant profile presented higher concentration for 100:0 and 0:100, but bovine milk has higher DPPH value, while during storage yogurts maintained constant values of DPPH, being higher for 0:100. In SEM and electrophoresis, it was observed that sheep milk has a more compact structure and smaller fat globules, higher concentration of proteins and their fractions than BM, with the mixture being 50:50 which kept during storage fractions of more compact caseins. We showed that yogurts containing a higher amount of sheep milk have potential higher nutritional value, showing the potential for application of this milk in the manufacture of yogurts and other dairy products.