Orientador: Prof. Dr. Flávio Faria de Moraes

Data da Defesa: 24/08/2012



 The use of enzymes in biotechnological processes is of great importance in the production of aromatic compounds, and the lipases are able to act on lipid substrate and catalyze hydrolysis reactions giving greater aroma intensity to dairy products. Considering this activity, the objective of this study was to develop butter formulations with the addition of microbial lipases from Thermomyces lanuginosus and Rhizopus oryzae and to check the effect of the hydrolysis of short chain fatty acids (up to 10 carbons) on aroma intensification. A 22 full factorial design was used for each lipase in order to determine enzyme concentration and time of hydrolysis. The cream was cooled to (5 ± 1) ºC for a period of 6 h and subjected to the churning process until phase inversion and formation of butter grains, which went through the steps of washing, pressing, weighing, salting, and division. The enzyme solution was added to the butter samples according to the proposed concentration for each microbial lipase. The product was subsequently packed in sterile packaging and incubated at 20 °C with enzymatic hydrolysis according to the time set in the factorial design. The formulations were sent for gas chromatography analysis and the effect of the variables on the hydrolysis process was analyzed. Statistical analysis was used to check the concentration of short chain fatty acids (mg/g of lipids) obtained in the butter formulations. Based on these results a hydrolyzed butter sample and the standard test were chosen to perform the physicochemical, instrumental, microbiological, and sensory analyses, and for comparison purposes, a commercial matured butter sample was also evaluated. Lipases were tested for hydrolytic activity and protein content. Cream, buttermilk, and butter formulations, called standard/control, hydrolyzed/lipolyzed and matured/commercial were subjected to physicochemical analyzes of total titratable acidity, pH, moisture and volatile solids, total solids, non-fat solids, ash, density, protein, free fatty acids, total lipid content,  chloride, peroxide value, saponification number, iodine index, diacetyl, and water activity. Instrumental analysis of fatty acids, texture, and color and microbiological analysis were performed to certify the quality of the produced butter. For sensory analysis, the three samples were evaluated by Hedonic Scale and Multiple Comparison tests by untrained and selected judges, respectively. Data was submitted to analysis of variance and Tukey studentized range test at 5 % probability. The results showed that cream and buttermilk had suitable composition for butter processing. As for the factorial design for Rhizopus oryzae lipase, the treatment using concentration of 45 mg/mL with 8 h of hydrolysis was the only one capable of reproducing the expected effects, differing from the standard sample in the sum of short-chain fatty acids. Enzyme concentration had a positive effect on the release of volatile fatty acids, indicating that the passage from the lower to the upper level resulted in an increase of 9.78 mg/g lipid. For the time factor, a negative effect on triacylglycerol hydrolysis was observed (-2.53 mg/g), which influenced the interaction of variables, with a decrease of 7.02 mg/g lipid during lipolysis. The prolonged incubation time also led to lower hydrolytic activity in preliminary tests with olive oil, showing values of 344 00 U/g in the first 5 min. As for butyric and caproic acids, no significant difference between treatments was observed. However, different results were obtained for caprylic and capric acids. For the lipase from Thermomyces lanuginosus, there was no significant increase in the amount of short chain fatty acids. However, the treated samples presented a strong smell of cheese, probably due to the significant amounts of hydrolyzed lauric acid. The analysis of effectsix showed that the increase in enzyme concentration led to a decrease in triacylglycerol hydrolysis, preventing the increase in the concentration of volatile fatty acids in the butter samples with this lipase. Therefore, the test with concentration of 45 mg/mL and incubation time of 8 h using Rhizopus oryzae lipase was selected. As for the physicochemical analyses, the highest averages for acidity (4.30 ± 0.03 %), free fatty acids (1.71 ± 0.01 %), and protein (0.58 ± 0.01 %) were observed for the lipolysed butter sample. Moisture content (16.55 ± 1.08 %) and chloride values (1.61 ± 0.01 %) were higher and pH (5.58 ± 0.03) was lower for the matured sample. Regarding lipids (83.88 ± 0.37 %), total solids (83.88 ± 0.60 %), non-fat solids (1.46 ± 0.24 %), and ash (2.09 ± 0.09 %), the standard formulation presented the highest values. As for iodine index the samples did not differ, but concerning saponification number the commercial sample presented the best values (161.05 ± 0.85). Peroxide and diacetyl were not detected in any of the evaluated formulations. Concerning water activity and consistency the commercial sample was statistically different from the others, with values of 0.93 ± 0.01 and 3281 gf/cm2, respectively. This sample also presented a
more yellowish color (b*= 34.69 ± 0.41). Considering fatty acids, the formulation with lipase showed higher amounts of short chain fatty acids (77.68 ± 2.71 mg/g), saturated fatty acids (618.76 ± 5.20 mg/g), and polyunsaturated fatty acids (10.36 ± 0.32 mg/g). Matured butter presented higher average values of monounsaturated fatty acids (341.41 ± 5.54 mg/g), which may have influenced the consistency results of this formulation. The samples submitted to microbiological analysis proved to be in accordance with legislation requirements and were sent for sensory evaluation. In the Hedonic Scale test, performed with untrained judges, the formulation with added Rhizopus oeyzae lipase attained the highest scores for taste (7.58 ± 1.12), aroma (7.35 ± 1.26), and color (7.52 ± 1.13), while in the evaluation with selected judges this same sample had higher scores for all attributes when compared with the standard formulation. These results are a good indicative of the importance of short chain fatty acids hydrolysis in enhancing butter aroma. In conclusion, the butter sample with added Rhizopus oryzae lipase at enzyme concentration of 45 mg/mL and hydrolysis time of 8 h presented higher amounts of short chain fatty acids than the standard and matured formulations, with higher scores in the sensory evaluation. Physicochemical analyses revealed good stability of the formulations and instrumental assessments indicated that matured butter was darker and with softened consistency, in accordance with the levels of  onounsaturated fatty acids found in this formulation. Keywords: Lipases, Aroma, Fatty acids, Hydrolysis