Nature-identical Compounds Research Articles

Stability of the antimicrobial activity of selected essential oils and nature identical compounds and their interaction with Tween 20 against reference bacterial strains of zootechnical interest

Essential oils (EOs) and nature identical compounds (NICs) express different antimicrobial activity (AAc) which can be affected by variability in composition, stability over time, and the carrier employed for their inclusion in feedstuff and products for animal treatments. The aim of the present study was to evaluate the stability over time of the AAc of 8 EOs and 5 NICs, alone and in combination with Tween 20, on four major bacterial livestock pathogens (E. coli, S. Typhimurium, S. aureus and MRSA). For this purpose, minimal inhibitory concentration (MIC) and checkerboard assay (CkA) tests were performed on EOs and NICs at batch first use (T0 – only MIC), and after 12 (T1) and 24 (T2) months. Several EOs showed initial MICs lower than 2% (v/v) and, except for clove bud and cinnamon EOs, reduced their efficacy over time. The best NICs’ AAc was showed at T1 by cinnamic aldehyde (against MRSA), carvacrol (against E. coli), and thymol (except against MRSA), while at T2 the MIC values decreased for most of the NICs. The CkA between EOs and Tween 20 showed different interactions (Fractional Inhibitory Concentration -FIC- index from 0.06 to 32.04) based on bacteria and time. No interactions were found between NICs and Tween 20 (FIC index from 1.0 to 3.0). The AAc of EOs and NICs changes over time. Therefore, the preservation interval of these additives is critical for the AAc of feedstuff and products intended for livestock animals. Moreover, the NICs seem more suitable for the association with Tween 20.

The use of thymol, carvacrol and sorbic acid in microencapsules to control Salmonella Heidelberg, S. Minnesota and S. Typhimurium in broilers.

The control of Salmonella spp. in poultry involves different biosecurity actions and lately has been complicated by the emergence of multidrug resistant serovars. The application of organic acids and essential oils has been used with different approaches due to the antibacterial properties as food preservatives. The use of these molecules in animal feed to control enteric pathogens is a major interest within the poultry industry. The use of a blend containing nature-identical compounds of sorbic acid (25%), thymol (9.5%) and carvacrol (2.5%) microencapsulated in a lipid matrix, was investigated in the present work, for the control of three Salmonella serovars (S. ser. Typhimurium, S. ser. Heidelberg and S. ser. Minnesota). Commercial broilers were challenged at 3 or at 33 days of age. Groups SH-1, SM-1 and ST-1, received treatment in the feed, at 2 kg/ton from 1-21 days of age and at 1 kg/ton from 35-42 days of age (last week), while groups SH-2, SM-2 and ST-2, were treated only during the last week receiving 2 kg/ton. Each treated group had an untreated control group, that was challenged at the same moment with the respective serovar (groups PCH, PCM and PCT). The challenge strains were enumerated in liver and cecal contents, weekly after challenge, at 7, 14, 21, 28, 35 and 42 days-of-age. Significant reduction was noticed at 7 and 14 days of age in all groups that received treatment during the initial phase (p < 0.05). Moreover, the body weight was significantly higher at the last experimental day (p < 0.05) in chickens that received treatment at the initial and at the final growth stages.

Antimicrobial Power of Organic Acids and Nature-Identical Compounds against Two Vibrio spp.: An In Vitro Study.

Vibrosis is one of the major threats in aquaculture farming, and due to the increasing antimicrobial resistance of different Vibrio species, there is an urgent need to replace conventional treatments with more sustainable solutions. Antimicrobial molecules such as organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in the infection management of terrestrial livestock, but little is known about their usage in aquaculture. The aim of this study was to perform a preliminary screening of the in vitro antimicrobial activity of a wide panel of OA and NIC against 2 Vibrio species characteristic of the Mediterranean area, Vibrio harveyi and Vibrio anguillarum, through minimal inhibitory/bactericidal concentration tests. The active principles that showed the best effective pathogen control were medium-chain fatty acids, sorbic and benzoic acid among OA and phenolic monoterpenoids (thymol, carvacrol and eugenol) and aromatic monoterpene aldehydes (vanillin and cinnamaldehyde) among NIC. These results showed how the usage of OA and NIC can open promising perspectives in terms of Vibrio spp. load control in aquaculture. Natural or nature-identical feed additives can make aquaculture production not only more efficient by reducing the need for medicated treatments, but also more sustainable.

Effects of dietary organic acids and nature identical compounds on growth, immune parameters and gut microbiota of European sea bass

A 71-day study was conducted to explore the effect of increasing dietary levels (0, 250, 500, 1000 mg kg feed−1; D0, D250, D500 and D1000, respectively) of a blend of microencapsulated organic acids (OA, specifically citric and sorbic acid) and nature identical compounds (NIC, specifically thymol and vanillin), on growth, intestinal immune parameters and gut microbiota (GM) of European sea bass juveniles reared under normal and subsequently suboptimal environmental conditions (high temperature, 30.0 ± 0.4 °C and low oxygen, 4.6 ± 0.6 mg L−1). OA and NIC did not promote growth, feed utilisation and feed intake at the inclusion tested but induced a significantly upregulation of IL-8, IL-10 and TGFβ. GM analyzed by next-generation sequencing showed that OA and NIC were able to exert prebiotic properties stimulating the development of beneficial bacteria taxa such as Lactobacillus, Leuconostoc, and Bacillus sp. Picrust analyses displayed a significant potential functional reconfiguration of GM promoting a decrease in inflammation-promoting and homeostatic functions at increasing OA and NIC administration. For the first time on this species the exposure to suboptimal rearing conditions was able to modify GM structure reducing LAB and increasing Proteobacteria, findings which were consistent with the inflammatory process observed at mRNA level.

163 AviPlus®S improves wean to finish performance in pigs

Abstract The study evaluated the effect of AviPlus®S, a microencapsulated blend of organic acids and nature identical compounds, fed to piglets from weaning through finishing. Ninety pigs (21 days of age, 6.47 ± 0.27 kg) were assigned to 3 diets (5 pigs/pen, n = 6) for a 22-weeks experimental period in 3 phases: weaning (weeks 1 to 6), growing (weeks 6 to 12), and finishing (weeks 12 to 22). The dietary treatments were: CTR, basal diet; AP1, basal + AviPlus®S at 1 kg/MT (weaning phase) and at 0.25 kg/MT (growing and finishing phase); AP2, basal + AviPlus®S at 2 kg/MT (weaning phase) and at 0.5 kg/MT (growing and finishing phase). Growth performance was recorded for each phase. Data were analyzed with ANOVA and differences considered significant at P&amp;lt; 0.05. In the weaning period, AviPlus®S linearly increased ADG and BW (P= 0.006 and P=0.003, respectively) with AP1 and AP2 pigs being heavier than control pigs by 0.44 and 1.1 kg, respectively, at week 6. Growing phase was not affected by treatments. In the finishing period AP2 had significantly higher ADG and G:F compared to control, while AP1 showed intermediate values (P=0.019 and P=0.005, respectively). Overall during the entire trial, AP2 pigs had the highest ADG and G:F, with the final BW being 5.6 kg higher than control at the end of the cycle (114.9 vs 109.3 kg).In conclusion, the supplementation with AviPlus®S allowed to improve growth performance of pigs throughout a production cycle from weaning to slaughter.

Effects of increasing dietary level of organic acids and nature-identical compounds on growth, intestinal cytokine gene expression and gut microbiota of rainbow trout (Oncorhynchus mykiss) reared at normal and high temperature

Organic acids (OA) and nature-identical compounds (NIC) such as monoterpenes and aldehydes are well-known growth and health promoters in terrestrial livestock while their application for fish production is recent and their mechanisms of action require further study. Hence, this study tested the increasing dietary level (D0, D250, D500, D1000; 0, 250, 500 and 1000 mg kg feed−1 respectively) of a microencapsulated blend containing citric and sorbic acid, thymol and vanillin over 82 days on rainbow trout to assess the effects on growth, feed utilization, intestine cytokine gene expression and gut microbiota (GM). Furthermore, the effects on intestinal cytokine gene expression and GM were also explored after one week at high water temperature (23 °C). OA and NIC improved specific growth rate (SGR) and feed conversion rate (FCR) during the second half (day 40–82) of the feeding trial, while at the end of the trial protein (PER) and lipid efficiency (LER) increased with increasing dietary level. GM diversity and composition and cytokine gene expression analysis showed no significant differences in fish fed with increasing doses of OA and NIC (82 days) demonstrating the absence of inflammatory activity in the intestinal mucosa. Although there were no statistical differences, GM structure showed a tendency in clustering D0 group separately from the other dietary groups and a trend towards reduction of Streptococcus spp. was observed in the D250 and D1000 groups. After exposure to high water temperature, lower GM diversity and increased gene expression of inflammatory intestinal cytokines were observed for both inclusions (D0 vs. D1000) compared to groups in standard condition. However, the gene up-regulation involved a limited number of cytokines showing the absence of a substantial inflammation process able to compromise the functional activity of the intestine. Despite further study should be conducted to fully clarify this mechanism, cytokines up-regulation seems to be concomitant to the reduction of the GM diversity and, particularly, to the reduction of specific lactic acid bacteria such as Leuconostoc. The application of the microencapsulate blend tested can be a useful strategy to improve growth and feed utilization in rainbow trout under normal temperature conditions. According to the results organic acids and nature-identical compounds did not revert the effects triggered by the increased temperature of water.

Nature-Identical Compounds and Organic Acids Ameliorate and Prevent the Damages Induced by an Inflammatory Challenge in Caco-2 Cell Culture.

Bioactive compounds, such as organic acids (OA) and nature-identical compounds (NIC), can exert a role in the protection of intestinal mucosa functionality due to their biological properties. The aim of this study was to understand the role of 2 OA (citric and sorbic acid) and 2 NIC (thymol and vanillin), alone or combined in a blend (OA + NIC), on intestinal barrier functionality, either during homeostatic condition or during an inflammatory challenge performed with pro-inflammatory cytokines and lipopolysaccharides (LPS). The study was performed on the human epithelial cell line Caco-2, a well-known model of the intestinal epithelial barrier. The results showed how OA and NIC alone can improve transepithelial electrical resistance (TEER) and mRNA levels of tight junction (TJ) components, but OA + NIC showed stronger efficacy compared to the single molecules. When an inflammatory challenge occurred, OA + NIC blend was able both to ameliorate, and prevent, damage caused by the pro-inflammatory stimulus, reducing or preventing the drop in TEER and improving the TJ mRNA expression. The data support the role of OA + NIC in modulating gut barrier functionality and reducing the negative effects of inflammation in intestinal epithelial cells, thereby supporting the gut barrier functionality.

Nature-Identical Compounds and Organic Acids Reduce E. coli K88 Growth and Virulence Gene Expression In Vitro.

Post-weaning diarrhoea (PWD) is one of the long-standing challenges in pig husbandry. Due to the risks of resistance caused by antibiotics (AB) misuse, conventional treatments against Escherichia coli K88 (E. coli K88), the PWD etiological agent, urgently need to be replaced. Organic acids (OA) and nature-identical compounds (NIC) are currently finding a central role in infection management thanks to their recognized antimicrobial activity. This study investigated the susceptibility of an E. coli K88 field strain to a wide panel of AB, NIC, and OA. Secondly, we evaluated the ability of sub-lethal doses of the most active compounds to modulate the expression of E. coli K88 virulence genes. Results showed that the bacterial strain was resistant to many of the tested antibiotics, but an antimicrobial action was registered for selected NIC and OA. The quantitative PCR analysis revealed that thymol, carvacrol, eugenol, and benzoic acid were able to downregulate (p < 0.05) the expression of bacterial genes related to motility, adhesion to enterocytes, heat-labile (LT) and heat-stable (ST) toxin secretion, quorum sensing, and biofilm formation. Therefore, this study demonstrated that selected OA and NIC not only control E. coli K88 growth but also modulate the expression of many virulence genes at sub-lethal doses, thus offering new insights on their mechanism of action and suggesting a powerful tool to manage PWD.

Thymol and Carvacrol Downregulate the Expression of Salmonella typhimurium Virulence Genes during an In Vitro Infection on Caco-2 Cells

Salmonella typhimurium is one of the major bacteria responsible for gastroenteritis in humans caused by foodborne pathogens. As pork is one of the main routes of transmission, bioactive compounds used as feed additives may be an important strategy to control Salmonella typhimurium. The aim of this study was to assess the antimicrobial activity of several organic acids and nature identical compounds against Salmonella typhimurium ATCC®® 6994™. Moreover, the effect of sub-lethal concentrations of thymol and carvacrol in counteracting a Salmonella typhimurium in vitro infection on Caco-2 cells was evaluated, focusing on the maintenance of the epithelial barrier and the alteration of Salmonella virulence genes. The results showed a protective effect of the compounds on the integrity of the intestinal monolayer, improving transepithelial electrical resistance and bacterial translocation compared to the non-treated cells. A real-time PCR study highlighted a significant downregulation of the main virulence genes of Salmonella (hilA, prgH, invA, sipA, sipC, sipD, sopB, sopE2). These findings indicate that thymol and carvacrol could be good candidates for the control of Salmonella typhimurium in pigs.

Single components of botanicals and nature-identical compounds as a non-antibiotic strategy to ameliorate health status and improve performance in poultry and pigs.

In the current post-antibiotic era, botanicals represent one of the most employed nutritional strategies to sustain antibiotic-free and no-antibiotic-ever production. Botanicals can be classified either as plant extracts, meaning the direct products derived by extraction from the raw plant materials (essential oils (EO) and oleoresins (OR)), or as nature-identical compounds (NIC), such as the chemically synthesised counterparts of the pure bioactive compounds of EO/OR. In the literature, differences between the use of EO/OR or NIC are often unclear, so it is difficult to attribute certain effects to specific bioactive compounds. The aim of the present review was to provide an overview of the effects exerted by botanicals on the health status and growth performance of poultry and pigs, focusing attention on those studies where only NIC were employed or those where the composition of the EO/OR was defined. In particular, phenolic compounds (apigenin, quercetin, curcumin and resveratrol), organosulfur compounds (allicin), terpenes (eugenol, thymol, carvacrol, capsaicin and artemisinin) and aldehydes (cinnamaldehyde and vanillin) were considered. These molecules have different properties such as antimicrobial (including antibacterial, antifungal, antiviral and antiprotozoal), anti-inflammatory, antioxidant, immunomodulatory, as well as the improvement of intestinal morphology and integrity of the intestinal mucosa. The use of NIC allows us to properly combine pure compounds, according to the target to achieve. Thus, they represent a promising non-antibiotic tool to allow better intestinal health and a general health status, thereby leading to improved growth performance.

Organic Acids and Nature Identical Compounds Can Increase the Activity of Conventional Antibiotics Against Clostridium Perfringens and Enterococcus Cecorum In Vitro

In a global context of increased antibiotic resistance, feed additives with enhanced antimicrobial properties are a useful and increasingly needed strategy. Organic acids (OA) and botanical molecules such as nature identical compounds (NIC) have been shown to be effective against bacterial infections based on their antimicrobial activity. The aim of this study was to evaluate whether the combination of OA or NIC with conventional antibiotics in poultry could increase antibiotic efficacy against Clostridium perfringens and Enterococcus cecorum . These organisms are the major poultry pathogens responsible for necrotic enteritis and bacterial chondronecrosis with osteomyelitis, respectively, and they have developed resistance to several antibiotics worldwide. A set of antimicrobial tests showed that both species had variable antibiotic sensitivity. Alternatively, OA and NIC were always effective in a dose-dependent manner, even when the antibiotics failed. For several strains, selected combinations of OA or NIC with antibiotics increased the bacterial sensitivity to antibiotics. Therefore, OA and NIC have potential to enhance the efficacy of conventional antibiotics against C. perfringens and E. cecorum .

Identifying Fraudulent Natural Products: A Perspective on the Application of Carbon-14 Analysis.

Across food and supplement industries, there is a growing trend toward products comprised of natural ingredients. As manufacturers strive to offer "natural" products to consumers, adulteration of ingredients with cheaper synthetic alternatives becomes a concern. This perspective highlights the application of carbon-14 analysis to screen for potential adulteration of natural ingredients, such as garlic oil. Carbon-14 testing determines if a product is comprised of solely plant- or animal-based ingredients by measuring the percentage of biomass versus petrochemical-derived sources. Through comparison to other analytical techniques used for quality control, carbon-14 testing stands out as being able to detect petrochemical-derived nature-identical compounds.

Microencapsulated sorbic acid and nature-identical compounds reduced Salmonella Hadar and Salmonella Enteritidis colonization in experimentally infected chickens

The reduction of Salmonella prevalence in broilers is a priority in European Union agricultural policies because treatment with antibiotics is forbidden by Regulation (EC) 2160/2003. Two trials were conducted to evaluate the efficacy of a microencapsulated blend of sorbic acid and nature-identical compounds (i.e., chemically synthesized botanicals; SAB) on the reduction of the cecal prevalence and contents of Salmonella enterica serovars Hadar and Enteritidis in experimentally infected chickens. In the first trial, 125 one-day-old Lohmann specific-pathogen-free chickens were assigned to one of the following treatments: negative control (not challenged and not treated), positive control (challenged and not treated), SAB0.3, SAB1, or SAB5 (challenged and treated with the microencapsulated blend included in the feed at 0.03, 0.1, or 0.5%, respectively). At 30 d of age, birds were infected with 10(6) cfu of Salmonella Hadar, and after 5, 10, or 20 d postinfection, 5, 10, and 10 birds per treatment, respectively, were killed and the cecal contents and liver and spleen samples were analyzed for Salmonella Hadar. In the second trial, 100 one-day-old Ross 708 chickens were assigned to 1 of 5 treatments: control (not treated), SAB0.3, SAB1, SAB2, or SAB5 (treated with the blend included in the feed at 0.03, 0.1, 0.2, or 0.5%, respectively). At 7 d of age, the birds were challenged with 10(5) cfu of Salmonella Enteritidis, and after 7, 14, or 24 d after challenge, 5, 5, and 10 birds per treatment, respectively, were killed and cecal contents were analyzed for Salmonella Enteritidis. Results showed that in the early stage of infection Salmonella prevalence was high in both studies, whereas at the end of the observation periods, the blends at 0.03, 0.1, and 0.5 in the challenge with Salmonella Hadar and at 0.2 and 0.5% in the challenge with Salmonella Enteritidis significantly reduced (by 2 log(10) cfu) the cecal content of Salmonella. This study showed that intestinal delivery of microencapsulated sorbic acid and nature-identical compounds can result in a 100-fold reduction of Salmonella at the intestinal level in broilers at slaughter age.

Feeding a microencapsulated blend of organic acids and nature identical compounds to weaning pigs improved growth performance and intestinal metabolism

One-hundred and seventy weaning pigs were divided in two groups, and housed in 34 pens (17 replicates, 5 pigs each). Piglets received a basal diet without (control diet, CTR) or with the addition of a microencapsulated blend of organic acids and nature identical compounds at 3 kg/ton (AVIplus®, EP1391155B1, Vetagro SpA, Italy). Piglets were weighed on d0, 14, and 41 of the experiment. Average daily gain and feed conversion rate were calculated between 0–14 d, 15–41 d, and 0–41 d. On d41, 8 animals per group were slaughtered and intestinal contents were collected to perform an analysis of microbial metabolites. Throughout the 0–41d period, AVIplus® group animals tended to have higher feed intake (+ 4.6%; P = 0.08), higher average daily gain (+ 8%; P < 0.01), and final body weight (+ 6.5%, P < 0.01). Tyramine was higher in AVIplus® group in cecum and colon, and spermidine and spermine resulted significantly lower in the stomach when compared to CTR. VFA was affected by AVIplus® all across the GI tract. Animals fed with AVIplus® had better growth performance, and a different metabolic intestinal pattern, due to the action of the released organic acids and nature identical compounds on microflora metabolism, being both polyamines and VFA as the end products of microbial fermentation.

The Online 18O/ 16O Analysis: Development and application

Oxygen is a globally important element, in inorganic compounds it is involved in key structural compounds of the earth, and it is providing functional groups for most organic compounds. Oxygen isotope ratio analyses on these compounds provide information on the oxygen sources and (formation) reactions in which the compound was involved. Therefore generally applicable analytical methods for the δ 18O determination are highly desirable. After a description of online high temperature "carbon reduction" methods for the conversion of oxygen in the compounds mentioned into CO and discussion of problems involved, especially in analysis of inorganic substances like carbonates and silicates, selected examples for the application of these carbon reduction methods will be presented. The well-known correlation of δ 18O values of cellulose (in tree rings) to that of (leaf) water is used since the 1970's as climate indicator. The observed enrichment in 18O in cellulose by about 27 in comparison to leaf water is attributed to an oxygen equilibrium isotope effect between carbonyl groups and water. But not all carbohydrates show this "general" 18O enrichment relative to water. By means of a recently published model a non-statistical oxygen isotope distribution in carbohydrates is postulated. Oxygen isotope discrimination has been recognized long ago as an important principle of authenticity evaluation in food and beverages. Though many basic mechanisms of isotope discrimination in nature are known, the corresponding application of data is often based only on empirical observations in this field. The fact that oxygen in natural organic plant material is derived from three sources (CO2, H2O, O2) with distinct differences in δ 18O and that their incorporation by different reactions implies oxygen isotope effects of different sizes, is therefore of special importance, and it is attempted by means of individual isotopic increments of functional groups to predict global δ 18O values of natural and nature-identical compounds. Vice versa δ 18O values can also give hints on the elucidation of biosynthetic pathways.

Synthesis and antispasmodic activity of nature identical substituted indanes and analogues

The synthesis of a series of substituted indanes which provide routes to nature-identical compounds and their analogues is reported. The smooth muscle relaxant activity of a series of substituted indanes and indanones together with their analogues has been measured. A compound with significant smooth muscle relaxant activity has been identified.