Título da Dissertação: Atividade antimicrobiana do Kombucha de chá verde fermentado em diferentes tempos frente a Alicyclobacillus spp.

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 20/02/2020


Alicyclobacillus spp. are gram-positive, spore-forming, non-pathogenic bacilli known to cause spoilage, such as undesirable odors and tastes in citrus-based foods. These bacteria are considered thermoacidophils because they are able to grow at low pH and high temperatures, so conventional heat treatments such as pasteurization become ineffective against these microorganisms. The use of natural antibacterial agents has been used as a sustainable alternative to problems related to antibiotic resistance and human irritation, as it has the ability to inhibit a wide range of microorganisms without compromising consumer health and environmental integrity. Studies show that Kombucha fermented beverage has antimicrobial activity against Gram-positive and Gram-negative microorganisms due to the bioactive compounds produced during fermentation.
The main objective of this research was to evaluate the antimicrobial capacity of the fermented drink Kombucha against Alicyclobacillus spp. Parallel to this, evaluate the antioxidant activity of the fermented beverage Kombucha and characterize it through the UHPLC-HRMS analysis.
Strains of Alicyclobacillus spp. from the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI), located at the Center for Chemical, Biological and Agricultural Research (CPQBA/UNICAMP) were used. The following microorganism species were used: A. acidoterrestris 0244T; A. herbarius 0246T; A. acidiphilus 0247T; A. cycloheptanicus 0297T and A. hesperidum 0298T. Kombucha crop was collected from a local grower in Maringá, Paraná and kept in green tea with sugar at different fermentation times 7 and 14 days. Antioxidant activity was determined by the free radical methods DPPH, ABTS and FRAP; Antibacterial activity was obtained from the analysis of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in 96-well microplates. Else, Kombucha samples were applied at concentrations of 1 ×, 4 ×, 8 × MIC in orange juice contaminated with A. acidoterrestris. Finally, the inoculum of A. acidoterrestris 0244T was treated with Kombucha samples fermented at different times at the concentrations defined by the sub MIC, MIC and the SEM (scanning electron microscopy) was held, antimicrobial activity to determine cell damage.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(CBM) of samples against bacteria A. acidoterrestris 0244T; A. herbarius 0246T; A. acidiphilus 0247T; A. cycloheptanicus 0297T, A. acidocaldarius 0298T yielded 50% (green tea), 1.553% (7-day fermented kombucha), 0.195% (14-day fermented kombucha) for all strains. While the minimum bactericidal concentration ranged from >50% to 12.5% for all isolates. The application of Kombucha at 1 ×, 4 ×, 8 × concentrations of the respective MICs in A. acidoterrestris contaminated orange juice (6.5 log) induced a reduction in colony count of up to 4 logs. Through the analysis of DPPH, FRAP and ABTS it was possible to verify that the fermented drinks have good antioxidant activity. The characterization of the samples by the UHPLC-HRMS analysis method showed that the fermentation of green tea with Kombucha increased the number of organic compounds in the drink, which may be responsible for the best result regarding antimicrobial activity. In addition, scanning electron microscopy (SEM) was able to show damage to cells treated with inhibitory concentrations of fermented Kombucha at different times.
Green tea kombucha fermented at different times showed effective inhibitory and
bactericidal activity at low concentrations against all evaluated Alicyclobacillus species. The results show that fermentation and its time affect the antimicrobial activity of Kombucha against spoilage bacteria, concluding that the longer the fermentation time the greater the antimicrobial activity. The fermented beverages showed good antioxidant activity and through the UHPLC-MS analysis, it was verified that the fermented green tea showed a higher number of metabolites compared to green tea without fermenting with symbiosis. Tests applied on the reconstituted orange juice proved to be effective because for both fermented beverages the concentration of 4 × MIC was sufficient to eliminate 4 logs of Alicyclobacillus acidoterrestris.

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