Artisanal beer production and evaluation adding rice flakes and soursop pulp ( Annona muricata L.)

study aimed to produce and evaluate the physicochemical characteristics of artisanal beers produced with the addition of rice flakes soursop pulp, and the acceptance level of these products sensory Abstract The possibility of varying beer formulations is acquired by variations in beverage production and the usage of different ingredients such as wheat, rice, corn, rye, cassava, honey, fruits, and others. Consequently, this work aimed to develop artisanal beers adding rice flakes and soursop pulp as adjuncts. Four artisanal beer formulations were elaborated, evaluating the following parametersI: °Brix, pH, density, alcohol content, total acidity, real extract, primitive extract, color and sensory acceptability. Results revealed that the samples were in accordance with the standards established by legislation. On the sensory analysis through the acceptance test, both pure malt beer and adjunct formulations exposed good acceptance in relation to the analyzed attributes. Regarding the beer formulations development, the results were satisfactory, ensuring quality artisanal beer varieties.

With the growing expansion of the beer market, special beers, known as artesanais (Dliveira & Silva, 2017;Tozetto et al., 2019). Artisanal beers are progressively gaining consumer preference, opening up competition and acquiring more space in the domestic market. Consumers are continusly looking for innovative and differentiated beers, returning the artisanal beer culture that began in the United States in the 1970s and intensified in Brazil in the 1990s with the incorporation of micro breweries that were after flavors innovation. Artisanal beer manufacture has gained strength and demonstrates in the economy to be a sector of great importance, generating various direct and indirect jobs (Madeira, 2015).
Considering these factors and also searching to encounter the beer consumers' new needs and preferences, the exploration for new formulations is justified with the addition of rice flakes and Brazilian tropical fruits such as soursop, so as to obtain a differentiated product for the consumer market. Therefore, this study aimed to produce and evaluate the physicochemical characteristics of artisanal beers produced with the addition of rice flakes and soursop pulp, and the acceptance level of these products through sensory analysis.

Soursop pulp obtaining
Soursop used in the preparations were harvested ripe directly from the tree, pulped and frozen. The fruits were thawed only the exactly quantities required to produce the beers.

Beer production
Four artisanal beer formulations were added with rice flakes and soursop with percentages established through preliminary testing using typical Ale beer formulations as displayed in Table 1, the ingredients were weighed separately.
Beer production, physicochemical analysis and sensory analysis were performed at the Food Production Laboratory, in the Chemical Technology Development Center (CDTEQ) Laboratory of the Universidade Estadual do Mato Grosso do Sul, Naviraí unity and Processing Laboratory of the Universdiade Federal de Grande Dourados.
Malts were obtained ready to start production, macerated by the company Brew Head Shop. On the mashing process, the Grain Bag, also known by the acronym BOAB (Brew in a Bag), was used. Ot is one of the simplest methods for artisanal beer brewing, mainly for the simplicity of the equipment (Palmer, 2006). Figure 1 presents the control artisanal beer production flowchart, the methodology used was according to Venturini (2010) with some adaptations.
The first process for beer production is mash. At this stage, malt and rice flakes were added to the equipment containing 10 L of mineral water, which was adjusted to the temperature of 68 ± 1 °C for 60 minutes. From this process, the must was obtained. After mashing, the filtration was started using a cake retention mesh, leaving only the liquid part in the equipment and the solid part in the mesh. On another equipment, 6.5 L of water at 78 °C was slowly dispensed under the cake. Subsequently the liquid part proceeded to the boiling step and the solid part was discarded.
After filtration, the must was warmed to boiling, and the bitterness hops were added starting the 60 minutes' count. After 5 minutes, the aroma hops were added. The must presents high temperature, so the equipment containing it was inserted into a basin immersed in ice and cold water with temperature around 0 °C, and the decantation occurred, aiming the sedimentation process at the bottom of the pan, also known as trub.
Must reaching the temperature of 20 °C, it was transferred to the fermenter and inoculated with high fermentation brewer's yeast of Saccharomyces cerevisiae and the equipment was closed. Ot was inserted in a greenhouse with temperature control at 18 ± 1 °C, initiating the fermentation, in which it remained for 7 days.
After seven fermentation days, the must was transferred by filtration to a container. For the formulations with the soursop pulp addition, it occurred at this stage. The bucket was sealed, starting the maturation stage, which lasted 10 days at temperature of 5 °C.
After the maturation process, the beers were filtered and filled in sanitized bottles containing 4 mL of priming (inverted sugar). Ot was then stored at room temperature for 10 days in order to occur the carbonation through the sugar fermentation by the action of remaining yeasts. Soon after carbonation, the beers were pasteurized in water bath at 62 °C for 30 min and stored at room temperature until the analyzes were performed.
All determinations were performed on the decarbonated samples. To remove CD 2 , the samples were transferred to 600 mL beakers and shaken with glass stick; the beer temperature was maintained at 20-25 °C until complete CD 2 removal (Onstituto Adolfo Lutz, 2008).

Sensorial analysis
For the sensory analysis, the 9-point hedonic acceptance test based on Dutcosky (2007) was applied; 1 corresponds to "very disliked" and 9 to "very much liked". Hedonic scales are applied in sensory analysis of different food matrices (García-Gómez et al., 2019;Ouliano et al., 2019). The following attributes were evaluatedI: color, bitterness, aroma, taste and overall impression. Sensory analysis was approved by the UEMS Human Research Ethics Committee under Dpinion nº 3.358.324.
Each taster received 30 mL of each beer sample at approximately 5 °C, coded with three random digits. Mineral water and salt and cracker were also served to clean the palate. The sensory panel was attended by 100 untrained judges of both sexes, aged from 18 years, being students, professors and employees of the Universidade Estadual de Mato Grosso do Sul -Naviraí Unit.

Statistical analysis
Physicochemical and sensory analyzes results were submitted to analysis of variance (ANDVA) and the means were compared by Tukey test with significance of 5%. Table 2 presents the physicochemical analyzes results performed on the samples. The °Brix mean values found in samples F2 and F3 differed from control and F1 samples statistically (p < 0.05), which can be attributed to the usage of different raw materials (rice flakes and soursop), resulting in higher values (6.9 and 7.0 °Brix). Current legislation does not determine a default value for this parameter, but comparing to other studies, it presented higher values than those encountered by Pereira & Leitão (2017), who analyzed pilsen artisanal beer and found values ranging from 5.3 to 6.0 °Brix.

Physicochemical analysis
The pH determination was performed in order to analyze its influence on the beer production process and to characterize the final product composition. All samples analyzed showed no significant difference (p < 0.05), displaying values below 4.5, being of fundamental importance, since it maintains the samples free of pathogenic microorganisms, and prevents further contamination (Hoffmann, 2001). The results were within the limit mentioned by Hardwick (1995), from 3.9 to 4.5 and Venturini & Cereda (2001), with pH values from 3.8 to 4.7 for pure malt beers.
Relative density values showed significant differences between samples (p < 0.05). According to Sousa (2009), the beer density determination allows the monitoring of alcoholic fermentation, because as yeast consumes sugars and produces alcohol, density values decrease, since sugars are denser than alcohol, or that is, for samples F2 and F3 which exhibited higher values, it can be said that both contained large sugars amounts in its compositions due to the added adjuncts (rice flakes and soursop).
Brasil (2009) determines as alcoholic beer the ones with alcoholic percentage superior than 0.5 ºGL in volume. The values obtained for the alcohol content were 4º GL for beer samples containing only malt in its formulation and 3º GL for samples containing rice flakes as adjunct. The lower F2 and F3 beers alcohol content may be related to the low obtainable fermentable sugars during gelatinization and hydrolysis of α-amylase flakes. This enzyme is characterized by producing from starch, mainly dextrins (non-fermentable) and peripherally glucose and maltose (Bamforth, 2003). Soursop addition in the maturation process did not interfere in the alcoholic fermentation, only conferred sensory characteristics to the beers. Schork (2015) obtained an alcohol content of 2.14 ºGL for beer with rice flour addition, which is lower than that obtained in this work.
According to Goiana et al. (2016), total acidity is related to total titratable organic acids in beer. The results found for this parameter are in accordance with the Brazilian legislation (Brasil, 2009), which establishes a variation of 0.1 to 0.3% acidity in beers. Brunelli et al. (2014) obtained an average of 0.28% total acidity for beer made with honey and Maia & Belo (2017) obtained an average of 0.26% for beer with soursop, showing that the values are in agreement with the present work.
Dne of the factors responsible for acidity is carbonic acid, resulting from the reaction between CD 2 and H 2 D, which may be responsible for the increase in acidity in the most carbonated beers. Most of the acids present in beer already exist in the mash, but in different proportions, and its concentrations vary 6.9 c ± 0.06 3.98 a ± 0.11 1.014 c ± 0.00 3.0 a ± 0.00 0.16 a ± 0.01 5.49 c ± 0.04 11.33 a ± 0.04 10.04ª ± 0.17 F3 7.0 c ± 0.06 3.90 a ± 0.08 1.015 d ± 0.00 3.0 a ± 0.00 0.18 b ± 0.01 5.59 c ± 0.04 11.42 a ± 0.04 20.14 b ± 0.57 Mean ± standard deviation. Control = Pure malt; F1 = Addition of 5% soursop; F2 = Addition of 40% rice flakes; F3 = Addition of 40% rice flakes and 5% soursop; SST = Total Soluble Solids. Equal letters in the same column are not significant by the tukey mean test (p < 0.05). EBC (European Brewing Convention).
depending on the raw material, malt variety and malting conditions (Venturini Filho,200). According to Sousa (2009), acidity in beer is important for its characterization, standardization and for the control of undesirable changes by microorganisms in the beer taste and aroma.
According to Brazilian legislation (Brasil, 1997), the real extract percentage indicates the amount of non-alcoholic ingredients that are found in beer after fermentation. Ot provides the body, color, foam stability and flavor. On beers, the real extract values must be between 2% and 7%; values above 3% are for good beer quality. Therefore, all samples presented good quality according to the obtained values, and the sample F3 obtained the greatest result. This is due to the ingredients in its composition, as rice flake has a large amount of non-fermentable sugars, and soursop was added only at the end of the process, giving only color and flavor to the beer.
For Brasil (2009), "[...] the primitive extract is the must substances (extract) amount that gave rise to beer, expressed as percentage by weight", being one of the usual classification forms; light beer rating 5 to 10.5%; beer 10.5 to 12.0%, extra 12.0 to 14.0% and strong > 14%. According to the results obtained, samples F1, F2 and F3 are classified as beer, and the control sample as extra beer, that is, the control formulation presented higher carbohydrates amount in the beer production, because in its formulation is used only malt that is rich in fermentable sugars.
Regarding color, beers with values below 20 European EBC (EBC) units are called light beers and for values above 20 EBC units, the beers will be classified as dark. Among the formulations, the only sample classified as light beer was F2, which had a value of 10.04 EBC units, while the other formulations were classified as dark beers. According to Venturini (2000), the assistant improves the beer physicochemical stability, reducing its turbidity. Ot is also responsible for giving the beer lighter color, as the pH reduction allows the loss of coloring substances. Table 3 presents the average sensory attributes result of the different beers with rice flakes and soursop additions. Attribute averages showed results within the product acceptance range, that is, between 6 and 7, ranging from "slightly liked" to "moderately liked".

Sensorial analysis
On the color sensory attribute, all formulations were well evaluated by the tasters, only the F1 formulation differed significantly (p < 0.05) from the other samples, probably due to a lighter coloration. Regarding the bitterness attribute, formulations F1 and F3 showed no significant difference.
For the sensory attribute "aroma", the F1 formulation presented the highest grade, but did not differ from the other formulations, indicating that the rice flakes and soursop addition pleased the tasters regarding this attribute. Formula F1 had a 6.65 grade for the "flavor" attribute, not statistically different from the control formulation that had the highest grade. This result indicates that the soursop addition pleased the tasters. Formulations F2 and F3 did not differ statistically from each other.
The scores for the F2 beer flavor and color attributes were higher than those found by Schork (2015) for gluten-free beer made with rice flour. According to the author the average grades were 3.18 (flavor) and 3.44 (color).
Regarding the attribute "Global impression" the formulations control and F1 were the ones that presented the highest scores and did not differ statistically from each other, also observing that there is agreement with the results of the sensory attributes "taste" and "aroma". However, formulation F1 also showed no significant difference between the other formulations F2 and F3, so overall all formulations showed good acceptance by the tasters.
New studies should be conducted using complementary sensory analyses, such as descriptive analyses in order to identify and quantify sensory descriptors (Torres et al., 2017) and also projective techniques, as a tool to study needs, feelings and motivations of purchasing behavior (Gambaro, 2018;Judacewski et al., 2019)

Conclusion
The beer samples were within the standards established by Brazilian law. On the physicochemical analysis, F3 beer (with rice flakes and soursop) presented a relative density of 1.015, alcohol content of 3.0 °GL, real extract of 5.59%, primitive extract of 11.42%, and the sensory analysis obtained values between 6.25-6.95 for the attributes analyzed in this work.
The addition of rice flakes and soursop as adjunct in brewing proved to be a viable alternative as it did not cause large variations in physicochemical parameters and was well accepted by the tasters.