Exploring the effect of a microencapsulated citrus essential oil on in vitro fermentation kinetics of pig gut microbiota.

Essential oils (EOs) have emerged as a potential alternative to antibiotics in pig breeding due to their antimicrobial properties. Citrus EOs, a common by-product of the orange juice industry, can be an interesting alternative from a financial perspective due to their huge offer in the global market. Thus, the effect of a citrus EO, and specifically different formulations of Brazilian Orange Terpenes (BOT), on pig gut microbiota was evaluated by means of an in vitro fermentation model simulating different sections of the pig gut (stomach, ileum, and colon). Treatments consisted in: BOT in its unprotected form (BOT, 1.85 and 3.70 mg/mL), microencapsulated BOT (MBOT, 3.50 and 7.00 mg/mL), colistin (2 µg/mL), and a control. BOT and MBOT altered in a similar way the total bacterial 16S rRNA gene copies in the stomach only from 18 h of incubation onwards, and no metabolite production in terms of short-chain fatty acids (SCFAs) was detected. In ileal and colonic fermentations, BOT and MBOT affected ileal and colonic microbiota in terms of total bacterial 16S rRNA gene copies, reduced phylogenetic diversity, and altered composition (p < 0.05) as evidenced by the significant reduction of certain bacterial taxa. However, more pronounced effects were found for MBOT, indicating its higher antimicrobial effects compared to the unprotected BOT, and suggesting that the antibacterial efficiency of the unprotected BOT was probably enhanced by microencapsulation. Furthermore, MBOT stimulated lactate production in ileal fermentations and greatly stimulated overall SCFA production in colonic fermentations. This indicates that besides the shifts in ileal and colonic microbiota by the delivered EO (BOT), the wall material of microcapsules (chitosan/modified starch) might have worked as an additional carbon source with prebiotic functioning, stimulating growth and metabolic activity (SCFAs) of colonic bacteria.

Essential Oils Extracted from Organic Propolis Residues: An Exploratory Analysis of Their Antibacterial and Antioxidant Properties and Volatile Profile.

The industrial processing of crude propolis generates residues. Essential oils (EOs) from propolis residues could be a potential source of natural bioactive compounds to replace antibiotics and synthetic antioxidants in pig production. In this study, we determined the antibacterial/antioxidant activity of EOs from crude organic propolis (EOP) and from propolis residues, moist residue (EOMR), and dried residue (EODR), and further elucidated their chemical composition. The EOs were extracted by hydrodistillation, and their volatile profile was tentatively identified by GC-MS. All EOs had an antibacterial effect on Escherichia coli and Lactobacillus plantarum as they caused disturbances on the growth kinetics of both bacteria. However, EODR had more selective antibacterial activity, as it caused a higher reduction in the maximal culture density (D) of E. coli (86.7%) than L. plantarum (46.9%). EODR exhibited mild antioxidant activity, whereas EOMR showed the highest antioxidant activity (ABTS = 0.90 µmol TE/mg, FRAP = 463.97 µmol Fe2+/mg) and phenolic content (58.41 mg GAE/g). Each EO had a different chemical composition, but α-pinene and ß-pinene were the major compounds detected in the samples. Interestingly, specific minor compounds were detected in a higher relative amount in EOMR and EODR as compared to EOP. Therefore, these minor compounds are most likely responsible for the biological properties of EODR and EOMR. Collectively, our findings suggest that the EOs from propolis residues could be resourcefully used as natural antibacterial/antioxidant additives in pig production.

Chemical Composition and Antibacterial and Antioxidant Activity of a Citrus Essential Oil and Its Fractions.

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.

Unraveling the selective antibacterial activity and chemical composition of citrus essential oils.

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.

Evaluation of the selective antibacterial activity of Eucalyptus globulus and Pimenta pseudocaryophyllus essential oils individually and in combination on Enterococcus faecalis and Lactobacillus rhamnosus.

Essential oils (EOs), as substitutes for antibiotics in animal diets, should have selective antibacterial activity between pathogenic and beneficial bacteria from the animal gut. Thus, this study evaluated the selective antibacterial activity of Eucalyptus globulus (EG) and Pimenta pseudocaryophyllus (PP) EOs on Enterococcus faecalis as a surrogate model of pathogenic bacterium and on Lactobacillus rhamnosus as a beneficial bacterium model. The EOs antibacterial activity was evaluated by determination of minimal inhibitory concentrations (MICs), minimal bactericidal concentration (MBCs), and fractional inhibitory concentration (FIC) indices. The time-kill and sequential exposure assays were also performed, but using only the EG oil, which was the best selective EO, since it had a MIC lower on E. faecalis (7.4 mg/mL) than on L. rhamnosus (14.8 mg/mL). FIC index values showed that the combination of the two EOs had an indifferent effect (1.25 and 2.03) on E. faecalis and an additive effect (1.00) on L. rhamnosus. The time-kill assay showed that EG oil was able to kill E. faecalis within 15 min of treatment (∼5 log reduction) and caused a reduction ∼3 log of L. rhamnosus viability. The sequential exposure assay showed that EG oil (at MIC/2) produced higher reduction on E. faecalis viability (∼3 log) than on L. rhamnosus (∼2 log) as well. Therefore, L. rhamnosus presented higher tolerance to the antibacterial activity of EG oil than E. faecalis did.