Development and Physicochemical, Sensory and Microbiological Characterization of Ferment Milk With Addition of Cupuaçu

The development of dairy products using probiotic microorganisms has been an important focus in the food industry. Thus, the present study aimed to evaluate the physicochemical, sensory and microbiological quality of fermented milk with addition of cupuaçu (Theobroma grandiflorum) flavor. The experimental design used was completely randomized, with five replicates, arranged in a 4 × 2 × 2 factorial scheme, consisting of fermented milk with addition of four different strains of probiotic bacteria (Lactobacillus casei shirota, Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus paracasei), presence and absence of cupuaçu flavor, and two evaluation times (1 and 7 days of storage). The results allowed us to conclude that the bacterial strain B (Lactobacillus casei) led to higher values of L*, b*, %Brix and pH. In the presence of cupuaçu flavor, the values of pH, L*, b* and %Brix were significantly higher, which allows us to infer that cupuaçu pulp contributes to decreasing the values of pH and increasing the acidity, lightness, b* and %Brix of fermented milk. Fermented milk with addition of bacteria A (Lactobacillus casei shirota) in the presence of cupuaçu flavor showed reduction in pH and %Brix and, therefore, better acceptance by consumers and bioconservation. The presence of cupuaçu flavor in fermented milk with bacteria B (Lactobacillus casei) alters L* and b* at seven days of storage. Fermented milk with addition of cupuaçu flavor constitutes an opportunity and possibility for developing new flavors from Amazonian fruits, strongly contributing to the durability of dairy products, since the physicochemical, sensory and microbiological characteristics are enhanced within the standards of food safety.


Introduction
Milk is a food composed of a combination of nutrients that are fundamental for consumption, such as water, carbohydrates, lipids, proteins, minerals and vitamins, such as A and D (fat-soluble) and B and C (water-soluble), which together provide important elements to health (Silva et al., 2008). Habitual consumption of this food is recommended, especially to achieve the recommended daily intake of calcium, a nutrient that, among other functions, is fundamental for the formation and maintenance of the body's bone structure (Muniz et al., 2013). Due to its nutritional profile, milk has been investigated in studies addressing the importance of its intake in the different phases of life, as well as potential health benefits attributed to its consumption and the use of probiotic bacteria.
The development of dairy products using probiotic microorganisms has been an important focus in the food industry. Currently, the production of foods containing specific probiotic strains with appropriate concentrations of viable cells along the shelf life is a technological challenge. Bacteria belonging to the genus Lactobacillus, such as those of the group Lactobacillus casei, Lactobacillus paracasei and Lactobacillus acidophilus, are very often used as probiotic supplements for food, especially for fermented milk, since they have been isolated from all portions of the gastrointestinal tract of healthy humans. The beneficial influence of probiotics on human intestinal microbiota includes factors such as antagonistic effects, competition and immunological effects, resulting in increased resistance against pathogens (Saad, 2006). Thus, the use of probiotic bacterial cultures stimulates the multiplication of beneficial bacteria, to the detriment of the proliferation of potentially harmful bacteria, reinforcing the natural defense mechanisms of the host (Puupponen-Pimiã et al., 2002). of milk pH, or reconstituted, with or without the addition of other dairy products, by lactic fermentation through the action of cultures of specific microorganisms, and these must be viable, active and abundant in the final product during its shelf life. Such fermentation is performed with one or more of the following cultures: Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium sp., Streptococus salivarius subsp. thermophilus and/or other lactic acid bacteria that, through their activity, contribute to determining the characteristics of the final product (Brazil, 2007). However, the production of fermented milk, such as fermented milk with addition of fruits, is a considerable alternative for dairy industries and can constitute a rational and logical form of different types of processing, with various flavors, being part of a promising market within the Brazilian scenario.
The Brazilian Amazon is rich in diversity of plants that are still little explored by most of the population and that can become a food alternative of high nutritional value and with alleged presence of nutraceutical compounds. Among the fruits of great importance for the Amazon region, cupuaçu (Theobroma grandiflorum) stands out as the most commercially exploited and is part of the group of tropical fruits with excellent commercial value. The pulp is the most consumed part of this fruit and, due to the characteristics of acidity, pectin content, active aroma, pleasant taste, high contents of volatile compounds and mineral salts (Martins, 2008), it is an important raw material for the processing industry. Data from the Food and Agriculture Organization (FAO) have shown that the worldwide marketing of fruit products has significantly grown in recent years. The use of fruit pulp in the formulations of dairy beverages offers some advantages, such as giving pleasant taste and contributing to improving the nutritional value of foods (Zulueta, 2007).
Several studies have been carried out in the different segments aimed at discovering new nutritional and functional sources, and the diversity of bioactive compounds in foods justifies the endless search for information on their physicochemical, physiological or functional and sensory properties. Thus, considering the potential of the Amazon region regarding the investment in food technology, the production of fermented milk with addition of Amazonian fruits creates the opportunity for discovering new horizons for products with different treatments and that meet both national and international consumers, through the creation and exploration of Amazonian products and species. In addition, research should be based on the fact that the strategic decisions to be made by the agents of the milk and dairy production chain depend on a deep understanding of consumer behavior, which has not yet been sufficiently investigated in the country.
In view of the above, the present study aimed to evaluate the physicochemical, sensory and microbiological quality of fermented milk with addition of cupuaçu (Theobroma grandiflorum) flavor.

Methods
The experiment was conducted at the premises of the Food Agroindustry of the Federal Institute of Education, Science and Technology of Rondônia, Colorado do Oeste Campus, in the municipality of Colorado do Oeste, RO, Brazil, at the geographic coordinates 13°06′ S and 60°29′ W, and average altitude of 407 meters.
The experimental design used was completely randomized, with five replicates, arranged in a 4 × 2 × 2 factorial scheme, consisting of fermented milk with addition of four different strains of probiotic bacteria (Lactobacillus casei shirota, Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus paracasei), presence and absence of cupuaçu flavor, and two times of evaluation (1 and 7 days of storage), in a total of 80 experimental units.
The milk used in the test was produced in the Animal Science sector of the Federal Institute of Education, Science and Technology of Rondônia, Colorado do Oeste Campus, and provided for the Food Agroindustry. Initially, this milk was skimmed to have a final maximum fat content of 0.5 g/100 g, according to the Normative Instruction nº 62 of 2011, in slow pasteurization, raising the temperature to 65 °C for thirty minutes, followed by cooling to the temperature of incubation of fermenting microorganisms (37 °C), in a rigorously controlled environment for a period of 7 hours. Milk fermentation occurred until the pH of the medium reached values of 4.0 (±0.25). This pH interval was determined considering the sensory and conservation characteristics of the product. The fermenting microorganisms used came from different commercial products and their samples were identified as treatments A, B, C and D, which correspond to: A (Lactobacillus casei shirota), B (Lactobacillus casei), C (Lactobacillus acidophilus) and D (Lactobacillus paracasei).
Fermented milk with and without cupuaçu flavor was produced by adding 9% sugar in each treatment. Subsequently, the milk was heated to prepare the syrup, and part of it received 5% of natural pulp of cupuaçu (Theobroma grandiflorum).
Treatments that, after processing, showed apparent viscosity visually similar to/compatible with that of fermented milk existing on the market were subjected to physicochemical analyses: pH, acidity, colorimetric parameters L* (lightness ranging from 0 [totally black] to 100 [totally white]), a * (-80 green to +100 red) and b * (intensity from -50 blue to + 70 yellow), and %Brix, analyzed at the first and seventh days of storage.
The sensory acceptance test (attributes: color, texture, consistency, aroma, taste, appearance and overall aspect) was performed at seven and fifteen days of storage, in triplicates. Sensory evaluation of acceptance was carried out by untrained tasters and considering the scores according to the hedonic scale with values from 1 to 9 points (1-disliked extremely, 2-disliked very much, 3-disliked moderately, 4-disliked slightly, 5-Neither liked nor disliked, 6-liked slightly, 7-liked moderately, 8-liked very much and 9-liked extremely), indicating how much they liked or disliked the color, texture, consistency, aroma, taste, appearance and overall aspect of the products. Purchase intention was evaluated by means of a mixed structured scale with 5 points (1-would buy, 2-possibly would buy; 3-maybe would buy, 4-possibly would not buy and 5-would not buy).
Microbiological determinations were performed according to the methodology described by Brazil (2003), evaluating the presence of total coliforms, Escherichia coli, Staphylococcus aureus, Salmonella sp., based on the microbiological parameters recommended by the legislation for fermented dairy beverages (Brazil, 2005).
The physicochemical, sensory and microbiological results were subjected to analysis of variance and the differences in the means were compared by Tukey test at 5% significance level, using Sisvar software.

Results and Discussion
The results showed significant effects (p ≤ 0.05) of the interaction between bacteria (B) and flavor (F) on b*, pH and %Brix (Table 1), while the interaction between bacteria (B) and time (T) influenced only the colorimetric parameter L* (Table 2), and the interaction between flavor (F) and time (T) only affected L* and b* (Table 3). There was no significant effect of the triple interaction of the factors on any of the physicochemical variables analyzed.
Further analysis of the double interaction between bacteria (B) and flavor (F) showed that the bacterial strain A was statistically superior to the probiotic bacteria B, C and D, both in the presence and absence of cupuaçu flavor for pH values (Table 1). In the presence of cupuaçu flavor, the bacterial strain A led to pH values on the order of 4.02, statistically differing (p≤0.05) from the treatment without cupuaçu flavor (pH of 4.32), which showed a 7.46% reduction in pH values in the presence of cupuaçu flavor and reduction in %Brix (Table 1). This probably occurred because of the low pH and high acidity of cupuaçu pulp. For the same analysis, bacteria D led to the lowest values of pH (pH 3.32) and, therefore, the highest values of acidity compared to the other bacteria (A, B and C), as well as the highest values of %Brix, both in the absence and presence of cupuaçu flavor (Table 1).
There was no statistical difference in pH values in the presence and absence of cupuaçu flavor for the treatment with bacteria D; the pH values remained stable, around 3.32, both in the absence and presence of cupuaçu flavor. In addition, there was no statistical difference in %Brix. Such higher acidity caused by bacteria D is related to the metabolites produced by these lactic acid bacteria, which contribute to inhibiting the growth of microorganisms. It is known that, at the beginning of fermentation, milk pH favors the development of Streptococcus salivarius subspecies thermophilus, which tends to be inhibited when pH reaches values from 4.2 to 4.4. However, the increase in acidification, that is, in lactic acid content, favors the growth of Lactobacillus delbrueckii subspecies bulgaricus, which can tolerate pH values within the range from 3.5 to 3.8 (Andrade, 2010). From the obtained data, it can be inferred that the pH values observed in the treatments meet the minimum value established for fermented milk recommended by the Technical Regulation of Identity and Quality (RTIQ) of Fermented Milks (Brazil, 2007).
The colorimetric parameter L* was higher in fermented milk with bacteria B in the presence of cupuaçu flavor, differing statistically from the other bacteria and in the absence of cupuaçu flavor. This means that the addition of bacteria B in milk fermentation promoted greater lightness in the product (Table 1), corroborating the data of the colorimetric variable b * , which pointed to a slightly yellowish color in the fermented milk, as shown in Table  2. There were significant effects of the different strains of probiotic bacteria at both times of storage of fermented milk on the colorimetric variables L* and b*. At seven days of storage, the bacteria B led to the highest values of L* and b*, compared to the other probiotic bacteria, and differed statistically (p ≤ 0.05) when compared to the first storage time (Table 2). At the first storage time, bacteria A differed significantly from the others, promoting the highest values of L* and lowest values of b* (Table 2).     Vol. 12, No. 5;2020 In all samples of fermented milk, the Most Probable Number/mL (MPN/mL) of coliforms at 35 °C and coliforms at 45 °C were lower than 0.3 MPN/mL. According to the RTIQ of Fermented Milk (Brazil, 2005), the MPN/mL of coliforms allowed for fermented dairy beverages is up to 100 MPN/mL of coliforms at 35 °C and 10 MPN/mL of coliforms at 45 °C for indicative samples. The results indicate adequate hygienic-sanitary quality throughout the process of production and storage of the fermented beverages and demonstrate microbiological quality above the level required by the current legislation, therefore suitable for consumption (Table 5). According to the Brazilian legislation, Salmonella spp. must be completely absent (Brazil, 2001). The shelf life of fermented milk was longer than 60 days, with maintenance of quality of the sensory characteristics of the product.
In the presence of cupuaçu flavor, the values of pH, L*, b* and %Brix were significantly higher, which allows us to infer that cupuaçu pulp contributes to decreasing the values of pH and increasing the acidity, lightness, b* and %Brix of fermented milk.
Fermented milk with addition of bacteria A (Lactobacillus casei shirota) in the presence of cupuaçu flavor showed reduction in pH and %Brix values and, therefore, better acceptance by consumers and bioconservation.
The presence of cupuaçu flavor in fermented milk with bacteria B (Lactobacillus casei) alters the colorimetric parameters L* and b* at seven days of storage.
The microbiological quality was higher than that required by the current legislation, indicating adequate hygienic-sanitary quality throughout the process of production and storage of the fermented beverages.
Fermented milk with addition of cupuaçu flavor constitutes an opportunity and possibility for developing new flavors from Amazonian fruits, strongly contributing to the durability of dairy products, since the physicochemical, sensory and microbiological characteristics are enhanced within the standards of food safety.