Contribution of environmental factors in the formation of biofilms by Alicyclobacillus acidoterrestris on surfaces of the orange juice industry / Contribuição de fatores ambientais na formação de biofilmes por Alicyclobacillus acidoterrestris em superfícies da indústria de suco de laranja

ABSTRACT: The objective of this study was to evaluate the effect of the initial microbial load, temperature and contact time on the biofilm formation of Alicyclobacillus acidoterrestris on stainless steel and natural food-grade rubber using orange juice as culture medium. The low initial load of A. acidoterrestris (2 log CFU/mL) led to biofilm formation on the stainless steel surface after 48 h of contact at 28 ºC and after 24 h at 45 ºC, and on natural food-grade rubber surface after 48 h of contact at both temperatures. The high initial microbial load (5 log CFU/mL) led to biofilm formation on stainless steel after 4 h of contact at 28 °C and 45 °C, while biofilm was formed on natural food-grade rubber after 8 h of contact at 28 °C and 4 h at 45 °C. The microbial load also affected the presence of spores in biofilm, which was observed on both surfaces only at high initial loads of A. acidoterrestris.

RESUMO: O objetivo deste estudo foi avaliar o efeito da carga microbiana inicial, temperatura e tempo de contato na formação de biofilme de Alicyclobacillus acidoterrestris em aço inoxidável e borracha natural de qualidade alimentar utilizando suco de laranja como meio de cultura. A baixa carga inicial de A. acidoterrestris (2 log UFC/mL) levou à formação de biofilme na superfície do aço inoxidável após 48 h de contato a 28 ºC e após 24 h a 45 ºC, e na superfície natural de borracha de qualidade alimentar após 48 h de contato nas duas temperaturas. A alta carga microbiana inicial (5 log UFC/mL) levou à formação de biofilme em aço inoxidável após 4 h de contato a 28 °C e 45 °C, enquanto o biofilme foi formado em borracha natural de qualidade alimentar após 8 h de contato a 28 °C e 4 h a 45 °C. A carga microbiana também afetou a presença de esporos no biofilme, o que foi observado em ambas as superfícies apenas com altas cargas iniciais de A. acidoterrestris.

Antibacterial activity of papain and bromelain on Alicyclobacillus spp.

Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 µg/mL and the MBC was 3.91 µg/mL, while theMIC of bromelain was 62.5 µg/mL and the MBCwas 250 µg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.

Evaluation of different culture media and enrichment in orange juice upon the growth of Alicyclobacillus spp / Avaliação de diferentes meios de cultura e enriquecimento em suco de laranja sobre o crescimento de Alicyclobacillus spp

Bacteria of the genus Alicyiclobacillus spp. form spores and develop in acid media, leading to the spoilage of citrus juices. Brazil is the largest exporter of orange juice concentrate, and yet, it has been extensively studied due to changes in taste and smell. Several investigations have reported different culture media used to detect and enumerate Alicyiclobacillus spp. The objective of the present study was to evaluate the recovery of Alicyiclobacillus spp. spores grown in ALI, BAT, K agar and YSG media using the methodology suggested by ABECitrus. Five inocula were used, two from reference strains and three from pasteurized concentrated orange juice. Cell recovery after the enrichment in reconstituted orange juice was also analyzed. An initial population of 6 log CFU/mL was inoculated. ALI, BAT and YSG media were able to recover the initial population of all different inocula, with no significant differences between the results. When compared to BAT, however, the preparation of ALI and YSG media was simpler and had more advantages. The recovery with K agar medium was lower than the other media for all the tested inocula, with significant differences found for Alicyclobacillus acidocaldarius0298T (3.66 log CFU/mL) and Alicyclobacillus pomorum-like CBMAI 0278 (4.11 log CFU/mL).(AU)

As bactérias do gênero Alicyiclobacillus spp. formam esporos e se desenvolvem em meios ácidos, podendo causar deterioração em sucos cítricos. O Brasil é o maior exportador de suco de laranja concentrado do mundo e, assim, este gênero vem sendo estudado por causar alterações de odor e sabor. Vários estudos relatam diferentes meios de culturas empregados para a detecção e enumeração de Alicyclobacillus spp. Este estudo teve como objetivo avaliar a recuperação de esporos de Alicyclobacillus spp. nos meios ALI, BAT, K ágar e YSG, utilizando a metodologia indicada pela ABECitrus. Cinco inóculos diferentes foram utilizados, sendo dois de linhagens-referência e os outros três isolados de suco concentrado de laranja pasteurizado. Também foi verificada a recuperação das células após o enriquecimento em suco de laranja reconstituído. Foi inoculada uma população inicial de 6 log UFC/mL. Os meios ALI, BAT e YSG conseguiram recuperar esta população nos diferentes inóculos, não existindo diferenças significativas entre os resultados. Contudo, devido à facilidade do preparo, os meios ALI e YSG mostraram-se mais vantajosos quando comparados ao meio BAT. O meio K ágar apresentou recuperação inferior aos outros meios para todos os inóculos, porém, houve diferença significativa apenas para Alicyclobacillus acidocaldarius 0298 T (3,66 log UFC/mL) e Alicyclobacillus pomorum-like CBMAI 0278 (4,11 log UFC/mL).(AU)

Evaluation of the antibacterial activity of Piperaceae extracts and nisin on Alicyclobacillus acidoterrestris.

Alicyclobacillus acidoterrestris is a gram-positive aerobic bacterium. This bacterium resists pasteurization temperatures and low pH and is usually involved in the spoilage of juices and acidic drinks. The objective of this study was to evaluate the antibacterial activities of nisin and the species Piper (Piperaceae) on A. acidoterrestris. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by the broth microdilution method. The species Piper aduncum had the lowest MIC and an MBC of 15.6 µg/mL and was selected for fractionation. Six fractions were obtained, and the dichloromethane fraction (F.3) had the lowest MIC/MBC (7.81 µg/mL). The dichloromethane fraction was again fractionized, and a spectral analysis revealed that the compound was prenylated chromene (F.3.7). The checkerboard method demonstrated that the crude extract (CE) of P. aduncum plus nisin had a synergistic interaction (fractional inhibitory concentration [FIC] = 0.24). The bactericidal activity of (F.3.7) was confirmed by the time-kill curve. P. aduncum, nisin, and prenylated chromene exhibited strong antibacterial activity against the spores and vegetative cells of A. acidoterrestris. The results of this study suggest that extracts of the genus Piper may provide an alternative to the use of thermal processing for controlling A. spoilage.

Resistance of Alicyclobacillus acidoterrestris spores and biofilm to industrial sanitizers.

This study evaluated the adhesion and biofilm formation of Alicyclobacillus acidoterrestris on industrial orange juice processing equipment and the bactericidal efficacy of peracetic acid, sodium hypochlorite, and quaternary ammonia after biofilm formation. The efficacy of these sanitizers against the spores of this microorganism was also evaluated. Stainless steel and nylon surfaces exhibited higher cell adhesion levels than did polyvinyl chloride surfaces. Peracetic acid was the most effective in removing biofilms from all surfaces (P < 0.05) and also reduced bacterial counts by 3 log CFU/cm² on the surface of polyvinyl chloride, but the other sanitizers also reduced the bacterial counts by 2 log CFU/cm². Quaternary ammonia exhibited the optimal minimum sporicidal concentration, preventing spore germination after only 15 s of contact at a concentration of 82 ppm. The flow cytometry results indicated that the spores and cells had low incidences of plasma membrane lysis after treatment with sanitizer, suggesting that lysis is not the principal mode of action for these sanitizers on A. acidoterrestris.