Post-weaning diarrhea (PWD) is an often disease affecting piglets. It is caused mainly by enterotoxigenic Escherichia coli (ETEC) colonization in pig gut. Antibiotics has been used to prevent, combat and control PWD and its negative impact on the productivity of pig breeding sector. Nonetheless, antibiotics due to their wide antibacterial spectrum also can reach beneficial gut bacteria, such as Lactobacillus. Lately, essential oils (EOs) have emerged as a potential alternative to using antibiotics in animal breeding because of their effect on bacterial growth. Commonly, citrus EOs are by-products of food industry and the availability of these EOs in the worldwide market is huge. Thus, six commercials citrus EOs were evaluated on ETEC strains, as model of pathogenic bacteria, and on Lactobacillus species, as models of beneficial bacteria. In overall, citrus EOs exhibited a selective antibacterial activity with higher effect on pathogenic bacteria (ETECs) than beneficial bacteria (Lactobacillus). Brazilian orange terpenes (BOT) oil presented the highest selective performance and caused higher disturbances on the normal growth kinetic of ETEC than on Lactobacillus rhamnosus. The action was dose-dependent on the maximal culture density (A) and the lag phase duration (λ) of the ETEC. The highest sub-inhibitory concentration (0.925 mg/mL) extended the λ duration to ETEC eight times (14.6 h) and reduced A in 55.9%. For L. rhamnosus, the λ duration was only extended 1.6 times. Despite the fact that limonene was detected as the major compound, the selective antibacterial activity of the citrus EOs could not be exclusively attributed to limonene since the presence of minor compounds could be implicated in conferring this feature.

Essential oils (EOs) from Citrus are the main by-product of Citrus-processing industries. In addition to food/beverage and cosmetic applications, citrus EOs could also potentially be used as an alternative to antibiotics in food-producing animals. A commercial citrus EO—Brazilian Orange Terpenes (BOT)—was fractionated by vacuum fractional distillation to separate BOT into various fractions: F1, F2, F3, and F4. Next, the chemical composition and biological activities of BOT and its fractions were characterized. Results showed the three first fractions had a high relative amount of limonene (≥10.86), even higher than the whole BOT. Conversely, F4 presented a larger relative amount of BOT’s minor compounds (carvone, cis-carveol, trans-carveol, cis-p-Mentha-2,8-dien-1-ol, and trans-p-Mentha-2,8-dien-1-ol) and a very low relative amount of limonene (0.08–0.13). Antibacterial activity results showed F4 was the only fraction exhibiting this activity, which was selective and higher activity on a pathogenic bacterium (E. coli) than on a beneficial bacterium (Lactobacillus sp.). However, F4 activity was lower than BOT. Similarly, F4 displayed the highest antioxidant activity among fractions (equivalent to BOT). These results indicated that probably those minor compounds that detected in F4 would be more involved in conferring the biological activities for this fraction and consequently for the whole BOT, instead of the major compound, limonene, playing this role exclusively.

Aim This study investigated the in vitro mechanism of action of a commercial citrus EO, Brazilian orange terpenes (BOT), on an enterotoxigenic Escherichia coli (ETEC) isolated from pig gut and on Lactobacillus rhamnosus. Methods and Results Firstly, bacteria were exposed sequentially to BOT every 3 h (three times) at sub‐MICs and results showed that sequential exposure to BOT provoked a higher reduction of bacteria viability than a single exposure and the reduction of ETEC viability was higher compared to that of L. rhamnosus. Then, evaluation of the BOT effects on the cell membrane permeability and integrity, indicated that BOT increased the membrane permeability and caused disruptive effects on the integrity of bacterial cells as reflected by an increase of the relative electric conductivity and the release of essential cell constituents. Interestingly, BOT effects were more pronounced on the ETEC than on L. rhamnosus. This was ratified by scanning electron microscopy (SEM), which showed more noticeable morphological damages and disturbances on ETEC cells than L. rhamnosus cells. Limonene was detected as the major compound in BOT by polar/non‐polar GC‐MS (78.65% / 79.38%). Conclusions Results revealed that the probable mechanism underlying the selective antibacterial action of the citrus EO, BOT, can be described as altering more remarkable the permeability and integrity of the cytoplasmic membrane as well as the external structure of an ETEC than L rhamnosus. Significance and Impact of the Study This study provides information about the mechanism of antibacterial action displayed by a citrus EO, a by‐product of the citrus processing industry, as a natural alternative to antibiotics used in pig production sector to combat pathogens as ETECs.