Título da Dissertação: Alicyclobacillus acidoterrestris: Resistência a sanitizantes industriais, multiplicação e produção de biofilme

Orientador: Prof. Dr. Benício Alves de Abreu Filho

Data da Defesa: 19/03/2013



 INTRODUCTION. Genus Alicyclobacillus spp. is composed of gram positive bacilli with cyclic fatty acids as the main component of the cell membrane. The forming spores are the main factors of resistance to the thermal processes in food. Growth pH ranges between 2.2 and 6.0 and temperature lies between 35ºC and 55ºC. Bacteria may change the sensorial characteristics of food, such as citric juices, since they produce guaiacol (2-methoxyphenol) providing unpleasant smell and taste to the products. These microorganisms are often associated to the degradation of reconstituted orange juice. In fact, it is unavoidable that fruits bear the bacterium or its spores to the respective industrial processes since the genus is widely distributed in the soil. Orange juice is the only Brazilian product that makes up 50% of world production and 85% of Brazilian exports. Consequently, it contributes significantly towards the country´s commercial balance; its deterioration brings high liabilities. Due to different pH bands and best temperatures for the growth of different species, it is currently difficult to identify a standard methodology for the development of Alicyclobacillus spp. In the case of bacteria control in the industrial juice processing, the industry employs procedures, such as fruit washing, aspersion by sanitizing products and pasteurization of the product. However, spores and biofilm resist all these procedures and the bacterium may nevertheless be present in the final product. Certain factors, such as concentration and contact time between sanitizing products and the microorganism, reduce the bacteria, but no conclusive data are extant on the activities of the sanitation products used in fruit washing on the spores and on the biofilm of Alicyclobacillus spp.
AIMS. Current experiment evaluates the recovery and growth of three species of Alicyclobacillus (A. acidoterrestris, A. acidocaldarius and A. pomorum-like) in the media currently employed in the literature: ALI agar (Alicyclobacillus medium), BAT (Bacillus acidoterrestris thermophilic agar), K-agar and YSG agar (Yeast extract starch glucose); evaluates the adherence and the formation of the biofilm of Alicyclobacillus acidoterrestris on the surfaces of the industrial processing of orange juice (stainless steel, PVC – polyvynil polychloride – nylon); evaluates the efficiency of sanitizing products such as peracetic acid, calcium hypochloride and quaternary ammonium in the removal of the biofilm and in the inactivation of the microorganism´s spores.
MATERIALS AND METHODS. Two strains (A. acidoterrestris DSM 3922T e A. acidocaldarius DSM 446T) were obtained from the German Collection of Microorganisms and Cell Cultures (DSZM – Deutsche Sammlung Von Mikroorganismen und Zellkulturen). Three species of Alicyclobacillus from the concentrated orange juice industries, identified and stored in the Brazilian Collection of Microorganisms of Environment and Industry (CBMAI, Campinas SP Brazil) were employed: A. acidoterrestris – CBMAI 0281; A. acidocaldarius – CBMAI 0294 e A. pomorum-like – CBMAI 0278. Culture media were prepared following legal protocols found in the literature, with pH for all media standardized to 4.0. Tested media were agar (Alicyclobacillus medium), BAT (Bacillus acidoterrestris thermophilic agar), K-agar and YSG agar (Yeast extract starch glucose). Tested surfaces for the production of biofilm were stainless steel coupons AISI 304 (1.0 x 1.0 x 0.1 cm), PVC – polyvynil polychloride (1.0 × 1.0 × 0.1 cm) and nylon bristles (1.0 × 0.1 X 0.1 cm), with surfaces washed one by one, hygienized and sterilized. Three chemical agents were selected: peracetic acid, sodium hypochloride and quaternary ammonium. Numbering of reference strains and isolates in the four media selected was undertaken by surface plating and incubation at 45ºC for 5 days. The numbering of Alicyclobacillus acidoterrestris with previous enrichment in reconstituted orange juice was also performed. A. acidoterrestris was inoculated in tubes with different surfaces to verify cell adherence to the surface. Treatment with sanitizing agents was them conducted. Quantification was undertaken by detachment of cells by sonication and plating. Adherence and sanitization were also assessed by scanning microscopy. Sporicide concentration for each sanitizing agent was also determined by microdilution. Flow cytometry with propidium iodide was performed to see whether sanitizing action affected the bacterium´s cell membrane. All assays were done in triplicate. Data were analyzed statistically by Assistat 7.6 Beta, while p<0.05 indicated significant difference.
RESULTS AND DISCUSSION. Plating in medium K agar in growth assays in culture media showed a low recovery of microorganisms when compared to the other three culture media. In the case of recovery of A. acidocaldarius and A. pomorum-like strains, significant differences existed for medium K agar when compared to other media but there was no significant difference between colony counts in different culture media for assays with A. acidoterrestris at 0h and after 24h of enrichment in juice at 45 °C. There was no significant difference between plating methods (surface and depth) employed in the assays. Characteristics in colonies of media ALI, BAT and YSG were similar, featuring circular, opaque colonies with regular light cream color margins, although YSG exhibited some slightly translucent colonies. The colonies in K agar medium had different morphological characteristics from other culture media, featuring colonies with irregular margins and some translucent colonies. In the case of adherence assays and biofilm formation, the biofilm in steel and nylon surfaces had a great cell adherence than that for PVC in all experimental conditions. Scanning electron microscopy showed the formation of a biofilm on the steel and nylon surfaces. PVC surface exhibited the adherence of cells and the production of extrapolysaccharides, although counts failed to supersede 5.0 Log UFC/cm2 after five days of incubation. When treatment with sanitizing agents on the surfaces after cell adherence is taken into account, all treatments showed significant differences from control, except treatment with 1000 ppm sodium hypochloride in PVC and nylon surfaces which failed to show any significant decrease of adhered cells. Moreover, 1000 ppm peracetic acid treatment had the highest efficiency since it reduced more than 2 Log UFC/cm2 in all surfaces. In the case of assays with spores, sanitizing agent with the highest sporicide activity was quaternary ammonium. Concentration 40.35 ppm completely inactivated the spores after a 10 min contact. Ammonium quaternary inactivated the spores and vegetative cells at low concentrations, although the latter were not efficient in the inactivation of A. acidoterrestris biofilm. Flow cytometry graphs suggest that the principal mechanism of sanitizing agents in the vegetative cell and in the spores of A. acidoterrestris do not occur because of membrane rupture since propidium iodide has a low fixation in the microorganism structures after treatment with sanitizing agents.
CONCLUSIONS. Comparisons between media ALI, BAT, K agar and YSG showed that media ALI, BAT and YSG recovered the initial population of different inoculums with no significant differences among results. Advantage lies in the easiness to prepare media ALI and YSG when compared to that of medium BAT. Medium K agar had a lower recovery for all inoculums, with significant differences for the recovery of Alicyclobacillus acidocaldarius CMAI 0298T and Alicyclobacillus pomorum-like CBMAI 0278. This fact shows that medium at pH 4.0 is not the best for the recovery of alicyclobacilli in orange juices with surface plating and incubation at 45°C for 72 to 120 h. Based on the results on cell amounts and scanning electron microscopy, it may be verified that adherence and formation of biofilm by A. acidoterrestris on the surfaces of stainless steel, PVC and nylon surfaces occurred. Sanitizing agents tested were not efficient in the total removal of biofilm cells of A. acidoterrestris for the evaluated conditions. Peracetic acid was the sanitizing agent with the highest reduction in cell numbers. In the case of all tested sanitizing agents, the double concentration, a bactericide for vegetative cells, only decreased approximately 2 Log UFC/cm2 after adherence and biofilm formation on the surfaces. The best sporicide activity occurred with quaternary ammonium which at low concentrations and contact time inactivated spores. Sanitization by peracetic acid and quaternary ammonium are highly efficient against Alicyclobacillus acidoterrestris for industries. However, they are not likely to be applied in the same hygienization process due to costs and practicability. For better results, recommended concentrations of sanitizing products used by the industry should be based on stricter conditions, taking into account the specific resistance mechanisms of each bacterium and of biofilm and spore formation.
Keywords: Alicyclobacillus, culture medium, spores, sanitizing agent, biofilm, orange juice.

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