Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 23/05/2022



INTRODUCTION: Among the microorganisms that represent a great concern in the food industry, Alicyclobacillus spp. strains stand out. They are non-pathogenic spore-forming bacteria that are related to the deterioration of drinks and citric juices. Among the 25 species of Alicyclobacillus that exist today, A. acidoterrestris is the most studied and challenging for the food industry since it alters products’ sensory characteristics. It is also the most isolated species in deteriorated and non-deteriorated sour products. Natural compounds as an alternative to replace synthetic chemicals in the food industry is something under frequent research regarding their possible application in food products. In their composition biologically active compounds with antimicrobial effects are present, especially in plants extracts, such as spices, herbs, fruits, and vegetables. Moreover, fruit by-products, such as pomace, peel, and seeds, have a series of bio-compounds already reported. Fruit by-products can often present high levels of bioactive compounds compared to their own pulp. Among these bio-compounds, the group of the phenolic and organic acids stands out with possible natural antimicrobial and antioxidant properties. Among the natural compounds we can mention kefir, which are grains constituted by polysaccharides in combination with a complex microbiota containing different lactic acid, acetic acid, bacteria and yeast. The metabolites produced by the fermentation of kefir, such as ethanol and organic acids, have antimicrobial activity against deteriorative and pathogenic microorganisms, such as Gram-positive and Gram-negative bacteria. In this manner, the use of fruit by-products rich in bioactive compounds as a substrate for kefir fermentation, is a strategy for obtaining products with higher levels of bioactive compounds and antimicrobial properties.
AIMS: The objective of this research was to evaluate the antimicrobial and antioxidant activity of different fruit by-products, such as grape, acerola and strawberry against different strains of Alicyclobacillus spp.
MATERIAL AND METHODS: Article 1: Four extracts were prepared with kefir grains, being 1 - grape extract and kefir grains; 2 - grape extract, brown sugar and kefir grains; 3 - grape extract, ultrasound and kefir grains; and 4 - grape extract, ultrasound, brown sugar and kefir grains. The four extracts were fermented at 28 ºC for 7 days. The extracts were centrifuged at 10,000 rpm for 10 min and the supernatant was subjected to membrane filtration (0.22 μm). The minimum inhibitory concentration (MIC) for A. acidoterrestris was determined by
the serial microdilution technique of extracts from 50 to 0.1% concentration in Bacillus acidoterrestris (BAT) medium. The minimum bactericidal concentration (CBM) was also determined. Structural changes in cells after treatment were evaluated by scanning electron microscopy (SEM). Article 2: 12 extracts were prepared using the subproducts of acerola, grape and strawberry without fermentation and after fermentation with Kefir for 24, 48 and 72 h. The extracts were centrifuged at 10,000 rpm for 5 min and the supernatant was submitted by membrane filtration (0.22 μm). The minimum inhibitory concentration (CIM) A. acidoterrestris 0244T, A. acidocaldarius subsp. rittmannii 0245T, A. herbarius 0246T, A. acidiphilus 0247T; A. cycloheptanicus 0297T, A. acidocaldarius 0299T, was determined by the serial microdilution technique of the extracts of 50 to 0.1% concentration in Bacillus acidoterretris (BAT) medium. The minimum bactericidal concentration (CBM) was also determined. For the MEV was used the A. acidoterrestris strain, and the inoculum was treated with the extracts of strawberry, grape and acerola fermented by 72 h. The antioxicant capacity of the extracts was measured through the methods of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Ferric Reducing Antioxidant Power (FRAP). And finally, the metabolites present in the extracts were identifier by UHPLCQtof- MS.
RESULTS AND DISCUSSION: Article 1: The MIC value capable of inhibiting the visible growth of A. acidoterrestris for all extracts was 1.6%, while the CBM was 50% for extracts 1 and 3, while for extracts 2 and 4 the CBM was 25%. The results show that extracts 2 and 4 obtained better CBM value, possibly because kefir produced more secondary metabolites with the addition of brown sugar, in addition, the use of ultrasound did not interfere. Article 2: The results show that extracts fermented for a longer period (72 h) had greater inhibition, and the extract of acerola by-product fermented for 72 h had the best results. For all strains, the Minimal Inhibitory Concentration (MIC) was 0.78%, except for A. acidocaldarius subsp. rittmannii, which obtained 1.56%. The same applied to the Minimal Bactericidal Concentration (MBC), in which 1.56% of the extract was capable of inactivating A. cycloheptanicus and A. acidocaldarius. The bioactivity of kefir drinks has already been studied, and demonstrated to contain substances with anti-inflammatory, antioxidant and antimicrobial activities. Other
authors analyzed the same fermentation times of kefir used in this study, however with kefir fermented in milk, and achieved a more efficient antibacterial activity from 36-48 h of fermentation against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa and Cronobacter sakazakii. Other studies also observed the action of kefir against yeast and pathogenic bacteria with the inhibition of Candida albicans, Salmonella Typhi, Shigella sonnei, E. coli and S. aureus by kefir fermented for 144 h. Although the present work investigated extracts of fruit fermented with kefir against Alicyclobacillus spp., the aforementioned studies corroborate our findings, since they indicate that antimicrobial activity increases with fermentation time, and it is attributed to the substances synthesized during the process. In addition, damages to the structure of the microorganisms caused by the extracts were verified by Scanning Electron Microscopy. Metabolite identification through liquid chromatography (UHPLC-Qtof-MS) demonstrated that the fermented extracts presented a greater number of compounds compared to the non-fermented ones, such as glucuronic, succinic and glutaric acids.
CONCLUSIONS: The results show that the by-products of fruits, fermented or not with Kefir, presented bioactive properties, such as antimicrobial potential against tested Alicylobacillus strains, and antioxidant potential, which results in an aggregate value product. In addition, our findings show an increase in antimicrobial activity with longer fermentation periods, with potential to be explored as an antimicrobial agent in the food industry. However, more research is needed to evaluate the use of such extracts in citrus drinks that can deteriorate due to the presence of Alicicylobacillus spp..
Key words: bio-compound; fermentation; antimicrobial compound; preservation;
deterioration, by-products.


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