Effect of a garlic and citrus extract supplement on performance, rumen fermentation, methane production, and rumen microbiome of dairy cows.

The aim of this trial was to determine the effect of a garlic and citrus extract supplement (GCE) on the performance, rumen fermentation, methane emissions, and rumen microbiome of dairy cows. Fourteen multiparous Nordic Red cows in mid-lactation from the research herd of Luke (Jokioinen, Finland) were allocated to 7 blocks in a complete randomized block design based on body weight, days in milk, dry matter intake (DMI), and milk yield. Animals within each block were randomly allocated to a diet with or without GCE. The experimental period for each block of cows (one for each of the control and GCE groups) consisted of 14 d of adaptation followed by 4 d of methane measurements inside the open circuit respiration chambers, with the first day being considered as acclimatization. Data were analyzed using the GLM procedure of SAS (SAS Institute Inc.). Methane production (g/d) and methane intensity (g/kg of energy-corrected milk) were lower by 10.3 and 11.7%, respectively, and methane yield (g/kg of DMI) tended to be lower by 9.7% in cows fed GCE compared with the control. Dry matter intake, milk production, and milk composition were similar between treatments. Rumen pH and total volatile fatty acid concentrations in rumen fluid were similar, whereas GCE tended to increase molar propionate concentration and decrease the molar ratio of acetate to propionate. Supplementation with GCE resulted in greater abundance of Succinivibrionaceae, which was associated with reduced methane. The relative abundance of the strict anaerobic Methanobrevibacter genus was reduced by GCE. The change in microbial community and rumen propionate proportion may explain the decrease in enteric methane emissions. In conclusion, feeding GCE to dairy cows for 18 d modified rumen fermentation and microbiota, leading to reduced methane production and intensity without compromising DMI or milk production in dairy cows. This could be an effective strategy for enteric methane mitigation of dairy cows.

Effects of dietary citrus extract on growth performance, carcass characteristics and meat quality of pigs.

This study investigated the effects of citrus extract on growth, carcass and meat quality of Duroc × Landrace × Large White pigs. One hundred and eight pigs (54 barrows, 54 females) were assigned to one of three dietary treatments for 138 days. The dietary treatments were (1) basic diet; (2) basic diet supplemented with 75 mg/kg chlortetracycline; and (3) basic diet supplemented with citrus extract (0.25 ml/kg during 56-112 days of age and 0.20 ml/kg during 113-194 days of age). No significant differences among treatments were found for growth performance, carcass characteristics, meat quality and free amino acids (p > 0.05). Feeding citrus extract tended to increase intramuscular fat (p = 0.052). Citrus extract and chlortetracycline increased C15:0 concentration (p = 0.016) and superoxide dismutase activity (p = 0.004). The pigs that received chlortetracycline exhibited the lowest (p = 0.033) muscle malondialdehyde concentration. Overall, citrus extract ameliorated some meat quality indicators without adverse effects on pig growth or carcass performance.

Dietary supplementation with citrus extract alters the plasma parameters, circulating amino acid profiles and gene expression of small intestinal nutrient transporters in Chinese yellow-feathered broilers.

BACKGROUND: This study evaluated the effects of citrus extract (CE) on growth performance, plasma amino acid (AA) profiles, intestinal development and small intestine AA and peptide transporter expression levels in broilers. A total of 540 one-day-old yellow-feathered broilers were fed a basal diet without any antibiotic (control group), or a basal diet containing 10 mg kg-1 zinc bacitracin (antibiotic group), or a basal diet supplemented with 10 mg kg-1 CE (CE group). After 63 days of feeding, two broilers per pen were slaughtered to collect tissues for further analysis. RESULTS: Results showed that CE increased (P < 0.05) the final body weight and average daily gain from day 1 to 63, and decreased (P < 0.05) the feed/gain ratio from day 1 to 63. Dietary CE supplementation increased (P < 0.05) plasma total protein, albumin and glucose concentration, and decreased (P < 0.05) urea concentration. CE supplementation increased (P < 0.05) the villus height in the ileum and the villus height/crypt depth in the jejunum and ileum, but decreased (P < 0.05) the crypt depth in the jejunum and ileum. CE supplementation increased (P < 0.05) most plasma essential AA concentrations. Additionally, CE supplementation upregulated (P < 0.05) ASCT1, b0,+ AT, B0 AT1, EAAT3, rBAT, y+ LAT2 and PepT1 expression in the jejunum, and b0,+ AT, EAAT3, rBAT, y+ LAT2, CAT1 and PepT1 in the ileum. CONCLUSIONS: Collectively, our results indicated that CE supplementation promotes intestinal physiological absorption of AAs by upregulating gene expression of small intestinal key AA and peptide transporters, thereby enhancing the growth performance of broilers. © 2020 Society of Chemical Industry.

Short communication: Effect of a citrus extract in lactating dairy cows.

Dry matter intake is a main driver of energy balance in lactating dairy cows, and some plant extracts have been commercially fed to dairy cows to stimulate feed intake. Citrus extracts contain several bioactive components and have been shown to modify metabolism in other animal models. Our hypothesis was that a citrus extract would increase dry matter intake. Two experiments were conducted to determine the effect of a citrus extract on intake and milk production in lactating dairy cows. In experiment one, 11 early-lactation dairy cows (experiment 1; 77 ± 15 d in milk, mean ± standard deviation) were used in a switchback design, and in experiment two, 15 mid-lactation Holstein cows (experiment 2; 157 ± 44 d in milk, mean ± standard deviation) were used in a crossover design. In both experiments, treatments were control (no supplement) or a citrus extract (4 g/d in experiment 1 and 4.5 g/d in experiment 2). Treatment periods were 21 and 14 d in experiment 1 and experiment 2, respectively, with the final 7 d used for sample and data collection. No effect was observed for treatment on dry matter intake, feeding behavior, milk yield, milk fat yield, milk protein yield, or milk composition in either experiment. Treatment also had no effect on milk trans fatty acid profile, but the extract increased total 16 carbon fatty acids 0.9 and 0.6 percentage points in experiment 1 and experiment 2, respectively. Plasma nonesterified fatty acids were decreased 6 h after feeding in both experiments (11.1 and 16.0 µEq/L in experiment 1 and experiment 2, respectively). Plasma insulin was increased 1 h before feeding compared with the control in experiment 1 (3.36 vs. 2.13 µIU/mL) and tended to increase 1.79 units 1 h before feeding in experiment 2. The citrus extract had no effect on feed intake or milk production at the dose investigated, but changed plasma insulin and nonesterified fatty acids, indicating some metabolic effects requiring further investigation.

Clinical study to assess the efficacy and safety of a citrus polyphenolic extract of red orange, grapefruit, and orange (Sinetrol-XPur) on weight management and metabolic parameters in healthy overweight individuals.

The present study investigated the efficacy and safety effects of Sinetrol-XPur (polyphenolic citrus dry extract) in weight management; metabolic parameters; and inflammatory, glycemic and oxidative status. In a 12-week, randomized, double-blind, placebo-controlled trial, Sinetrol-XPur was given to overweight subjects twice daily with meals in the tested group (N = 47) versus a placebo group (N = 48). Waist and hip circumference and abdominal fat were decreased in the Sinetrol-XPur group as compared with the placebo group (p < 0.0001) (-5.71% vs. -1.56% for waist, -4.71% vs. -1.35% for hip and -9.73% vs. -3.18% for fat). Inflammatory markers were reduced (C-reactive protein: -22.87% vs. +61%; fibrinogen: -19.93% vs. -1.61%, p < 0.01). Oxidative stress was lowered as seen by the reduction of malondialdehyde (-14.03% vs. 2.76%) and the increase in superoxide dismutase and glutathione (17.38% vs. 2.19% and 4.63% vs. -2.36%, respectively, p < 0.01). No adverse effects were observed. Kidney, liver, and lipid panels remained unchanged. These results indicated that Sinetrol-XPur supplementation is a viable option for reducing abdominal fat, waist and hip circumference, and body weight and for improving inflammatory, glycemic, and oxidative status in healthy overweight individuals.

Effect of a Garlic and Citrus Extract Supplement on the Lactation Performance and Carbon Footprint of Dairy Cows under Grazing Conditions in Chile.

Two trials were conducted to evaluate the effect of a garlic and citrus extract supplement (GCE) on the milk production performance and carbon footprint of grazing dairy cows in a Chilean commercial farm. A total of 36 early- to mid-lactation and 54 late-lactation Irish Holstein-Friesian cows were used in Trial 1 and Trial 2, respectively. In both trials, the cows were reared under grazing conditions and offered a supplementary concentrate without or with GCE (33 g/cow/d) for 12 weeks. The concentrate was fed in the afternoon when the cows visited the milking parlour. Consequently, the results of milk production performance in these trials were used to determine the effect of feeding with GCE on the carbon footprint (CFP) of milk using a life cycle assessment (LCA) model. In Trial 1 and Trial 2, feeding with GCE increased estimated dry matter intake (DMI, kg/d) by 8.15% (18.4 vs. 19.9) and 15.3% (15.0 vs. 17.3), energy-corrected milk (ECM, kg/d) by 11.4% (24.5 vs. 27.3) and 33.5% (15.5 vs. 20.7), and feed efficiency (ECM/DMI) by 3.03% (1.32 vs. 1.36) and 17.8% (1.01 vs. 1.19), respectively. The LCA revealed that feeding with GCE reduced the emission intensity of milk by 8.39% (1.55 vs. 1.42 kg CO2-eq/kg ECM). Overall, these results indicate that feeding with GCE improved the production performance and CFP of grazing cows under the conditions of the current trials.

Fine characterization and microbiota assessment as keys to understanding the positive effect of standardized natural citrus extract on broiler chickens.

The objective of this study was to investigate the effect and composition of a standardized natural citrus extract (SNCE) on both broiler chickens' growth performances and intestinal microbiota. A total of 930 one-day-old males were randomly assigned to three dietary treatments: a control treatment (CTL) in which broiler chickens were fed with a standard diet and two citrus treatments in which broiler chickens were fed with the same standard diet supplemented with 250 ppm and 2,500 ppm of SNCE, respectively. Each dietary treatment was composed of 10 experimental units (pen) of 31 broiler chickens each. Growth performances such as feed consumption, body weight, and feed conversion ratio (FCR) were recorded weekly until day 42. Litter quality was also weekly recorded while mortality was daily recorded. One broiler chicken was randomly selected from each pen (10 chickens/group) and ceca samples were collected for microbiota analysis at day 7 and 42. Chromatographic methods were used to determine molecules that enter into the composition of the SNCE. Results from the characterization of SNCE allowed to identify pectic oligosaccharides (POS) as a major component of the SNCE. In addition, 35 secondary metabolites, including eriocitrin, hesperidin, and naringin, were identified. The experiment performed on broiler chickens showed that the final body weight of broiler chickens fed diets supplemented with SNCE was higher than those fed the CTL diets (P < 0.01). Broiler cecal microbiota was impacted by age (P < 0.01) but not by the dietary supplementation of SNCE. Results indicate that SNCE allowed enhancing chickens' performances without any modulation of the cecal microbiota of broiler chickens. The characterization of SNCE allowed to identify compounds such as eriocitrin, naringin, hesperidin, and POS. Thus, opening new horizons for a better understanding of the observed effect on broiler chickens' growth performances.

Citrus extracts are increasingly being used in animal nutrition to enhance animal growth performances. Most of the available studies indicate an effect of these extracts on microbiota. However, citrus extracts can vary a lot. Indeed, the composition of citrus extract depends on parameters such as the citrus species, the extraction methods, and the inclusion rate. This variation is very important to take into consideration before using a citrus extract. The objective here was to evaluate a commercially available standardized natural citrus extract in terms of composition and effect on broiler chickens' performances and microbiota. Results showed that standardized natural citrus extract positively affects the final weight of broilers, but no effect was observed on chickens' caecal microbiota. The characterization of the standardized natural citrus extract reveals pectic oligosaccharides as major compounds as well as 35 others molecules. Most of these compounds are well described for their beneficial effect on animals' performances and health. In conclusion, the standardized natural citrus extract showed beneficial effects on broilers' performances. These effects are not correlated with broilers microbiota modulation and may be explained by the composition of the product.

Mitigating the Environmental Impacts from Pig and Broiler Chicken Productions: Case Study on a Citrus Extract Feed Additive.

The rapid expansion of the livestock production sector to meet the world population's demand is posing a big challenge to environmental sustainability. Plant-based feed additives extracted from agro-food byproducts could potentially result in multiple outcomes: reducing food-processing wastes and improving animal growth performances, hence mitigating environmental impacts of meat production chains. This presented study was carried out to assess the environmental impacts of the use of a commercial citrus extract feed additive (CEFA) in swine and broiler chicken farming. Life-cycle assessment (LCA) was applied to assess the impact of manufacturing and distributing one 25 kg bag of CEFA and its use in feed in broiler chicken and swine productions. With regards to CEFA manufacturing and distribution, results showed that most of the impact came from the production of CEFA ingredients, accounting for 70% of the impact generated. The remaining 30% effect was divided between transportation to the customer (25%), CEFA packaging (3%), and CEFA manufacturing and production loss (2%). When enlarging the scope, the use of the CEFA in pigs and broilers' diets was shown to improve the measured environmental indicators, compared to such standard systems. Indeed, CEFA-added feeds have demonstrated enhanced growth performances, hence reducing the required amount of consumed feed to achieve the same level of growth. Consequently, this helped reduce environmental issues from animal feed ingredients' agriculture. To be more specific, the use of one 25 kg bag of CEFA in feed at 250 g per ton of feed led to a reduction of 6 tons of CO2 equivalent (CO2 eq) emitted along the life cycle of poultry production and 5 tons in the case of fattening pigs. The inclusion of this CEFA in the diet also led to a reduction in the land use footprint by 0.7 hectares and reductions in water consumption by 201 m3 and 82 m3 for broiler chicken and swine production, respectively. The environmental performance assessment thus showed the interest in using this CEFA in swine and broiler chicken diets to mitigate the environmental impacts.

Citrus Extract High in Flavonoids Beneficially Alters Intestinal Metabolic Responses in Subjects with Features of Metabolic Syndrome.

The objective of this study was to investigate the effects of a citrus extract rich in citrus flavonoids on intestinal metabolic responses in subjects with features of metabolic syndrome, in an in vitro colon fermentation system (TIM-2) and fecal samples obtained from human subjects in an in vivo trial. In the TIM-2 system inoculated with fecal samples of volunteers with features of metabolic syndrome, continuous citrus extract supplementation (500 mg/day) resulted in increased cumulative short-chain fatty acid (SCFA) levels compared to the control condition, which was mainly due to increased production of butyrate, acetate, and valerate. In human volunteers, 12 weeks of daily supplementation with 500 mg citrus extract resulted in a significant shift in the SCFA profile towards more butyrate (p = 0.022) compared to the placebo group. Furthermore, there was a trend towards a reduction in fecal calprotectin levels, a marker for intestinal inflammation, compared to the placebo (p = 0.058). Together, these results suggest that citrus extract intake may have a positive effect on intestinal metabolic responses and through this, on host health in subjects with features of metabolic syndrome. Further research is needed to provide more insight into the potential underlying mechanisms and to study effects on clinical parameters.

Lipidomic profiling using GC and LC-MS/MS revealed the improved milk quality and lipid composition in dairy cows supplemented with citrus peel extract.

Eight lactating cows were used to determine the effects of citrus peel extract (CPE) on milk performance, antioxidant properties, and milk lipids composition. CPE supplementation up to 150 g/d (CPE150) increased milk yield and the proportions of unsaturated fatty acids of conjugated linoleic acid. CPE with abundant polyphenol and flavonoids can transfer these bioactive substances to mammary gland and improve the antioxidant properties of milk obtained from cows. Lipidomics revealed that 56 lipid species were altered between CON vs CPE150, and there were five key differential metabolic pathways. In particular, milk phosphatidylethanolamine and phosphatidylcholine were significantly increased with dietary CPE supplementation. In summary, our results provide insights into the modifications in the milk components and milk quality of dairy cows received CPE. The inclusion of CPE in the diet of dairy cows may be an effective and natural way to increase the antioxidant amounts and beneficial lipids in milk.

Mixture design methodology and predictive modeling for developing active formulations using essential oils and citrus extract against foodborne pathogens and spoilage microorganisms in rice.

The antibacterial and antifungal effects of six plant-derived essential oils (EOs) and two types of citrus extracts (CEs) were studied against two pathogenic bacteria (Salmonella Typhimurium and Escherichia coli O157:H7) and three fungi (Aspergillus niger, Penicillium chrysogenum, and Mucor circinelloides). A broth microdilution assay and checkerboard method were used to measure the minimal inhibitory concentration (MIC) of each extract and the possible interactions between them. The MIC assay showed that cinnamon EO, Mediterranean EO, Southern formulation, citrus EO, organic citrus extract (OCE), and natural citrus extract (NCE) had the highest antimicrobial and antifungal activity. The checkerboard method showed that the Mediterranean EO+OCE combination acted in synergy against all tested pathogens. A centroid mixture design was used to develop active formulations by predicting optimal concentrations of EO/CEs for increased antibacterial/antifungal activity. A mixture of four formulations (625 ppm OCE, 313 ppm Mediterranean EO, 625 ppm citrus EO, and 313 ppm cinnamon EO) named as active formulation 1, and the mixture from five formulations (625 ppm NCE, 625 ppm Asian formulation, 313 ppm Southern formulation, 625 ppm cinnamon EO, and 313 ppm savory thyme EO) named as active formulation 2, were formulated and tested because of their high microbicidal effectiveness. In situ tests with rice showed a significant reduction (P ≤ 0.05) of all tested pathogenic bacteria and fungi from the vapor of active formulations 1 and 2 after 28 days of storage. PRACTICAL APPLICATION: Active formulations (essential oils and citrus extracts) developed in the study are highly effective against foodborne pathogens. Active formulations in this study could be used as natural preservatives in the food industry for controlling foodborne diseases and spoilage organisms in stored foods.

Antiseptics and the Ocular Surface: In Vitro Antimicrobial Activity and Effects on Conjunctival and Corneal Epithelial Cells of a New Liposomal Ocular Spray Containing Biosecur® Citrus Extract.

INTRODUCTION: The study aimed to evaluate the in vitro antimicrobial activity of a new liposomal ocular spray containing the antiseptic Biosecur® citrus extract (Oftasecur, OFFHEALTH, Florence, Italy) and its in vitro effects on cultured human corneal and conjunctival cells. METHODS: Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of Oftasecur against Candida albicans and Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains, were determined. Human corneal and conjunctival epithelial cells in vitro were incubated for 10 and 30 min with Oftasecur or its components. The cytotoxicity was assessed through the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability. RESULTS: Oftasecur was active at dilutions ranging from 1:2 to 1:16 and it displayed bactericidal and fungicidal effect against all assayed microorganisms. Most of the reduction of Staphylococcus epidermidis vitality (65%) occurred within the first minute of exposure. The cytotoxicity of Oftasecur was similar to its vehicle, and the cell viability was significantly reduced only by Oftasecur in its undiluted form. Conversely, Biosecur induced a significant cytotoxicity in all the experiments. CONCLUSION: Oftasecur showed a rapid and wide-spectrum antibacterial activity, with an optimal in vitro tolerability profile.

Impact of a Dietary Citrus Extract on the Behavior and Production of Lactating Dairy Cows Following Regrouping: A Preliminary Study.

Exposure of lab animals, humans, and pigs to olfactory sensory feed additives may reduce response to stress and anxiety. The objective of this preliminary study was to determine if feeding a citrus-based olfactory sensory functional feed extract (derived from Citrus sinensis) reduces the negative impact of regrouping of lactating dairy cows. Thirty-two (parity = 2.0 ± 1.2; mean ± SD), mid-lactation Holstein dairy cows (169.8 ± 16.8 DIM) were enrolled as focal cows in this study and housed individually in a tie-stall facility where they were randomly assigned to 1 of 2 treatment diets: (1) control total mixed ration (TMR) (control; n = 16; primiparous = 7; multiparous = 9), or (2) control TMR with 4 g/d of citrus extract (CE) (Phodé, Terssac, France) (CE; n = 16; primiparous = 7; multiparous = 9). Cows were fed their experimental diets for 7 d in the tie-stall facility (baseline), then moved to 1 of 2 experimental free-stall pens (containing 29 other cows) for a period of 7 d, where they remained on the same treatment diet as before. Compared with their baseline, primiparous control cows had decreased rumination time on d 1 and 2, had decreased lying time on d 1, and tended to have decreased lying time on d 2 and 3 following regrouping. In contrast, primiparous cows fed the CE diet did not experience a change in rumination and lying time. Primiparous CE cows had greater feeding time on d 1 and tended to have greater feeding time on d 2 after regrouping compared to primiparous control cows. Primiparous control cows had greater idle standing time, as compared to the CE cows, across the 7 d after regrouping. Primiparous CE cows initiated less total competitive behavior after regrouping, but were also displaced more frequently from the feed bunk and from the free stalls on d 1 after regrouping, as compared to the primiparous control cows. For multiparous cows, CE supplementation was not consistently associated with any benefits to behavior or production after regrouping, possibly because these cows were more experienced with social stressors. Results indicate that feeding CE to mid-lactation naïve primiparous dairy cows may reduce the initiation of competitive interactions and lessen the reduction in rumination and lying time after regrouping. These results need to be verified in further studies where potential confounding effects (e.g., pen social dynamics, pen location) are minimized.

Standardized Natural Citrus Extract dietary supplementation influences sows' microbiota, welfare, and preweaning piglets' performances in commercial rearing conditions.

We investigated the effect of the Standardized Natural Citrus Extract (SNCE; Nor-Spice AB, Nor-Feed SAS, France) on the microbiota of the sows and on the weight gain of their piglets. Fifty sows were randomly divided into two groups: a control group (23 sows) with a standard diet and a SNCE group (27 sows) with a standard diet supplemented with 2,500 ppm of SNCE. Supplementation occurred 10 d before and 5 d after farrowing. Fecal samples from 16 sows (8 randomly selected sows of each dietary treatment) were collected for the fecal microbiota analysis 5 d after farrowing. The supplementation of SNCE increases the amount of cultivable Lactobacillus threefold in vitro. Microbial DNA was extracted from the fecal samples for sequencing of the 16S rRNA gene. The SNCE, which affected the microbiota as a discriminant analysis, was able to separate the microbial communities of the eight sows that received SNCE from the three control sows with 21 Operational Taxonomic Units (area under the ROC curve = 96%). SNCE also reduced the interval between farrowing and the first dejection of the sow and increased their feed intake (P-value < 0.05). Furthermore, feeding the sows with SNCE improved the weight gain of the piglets in the first week of life. These results show that SNCE supplementation allows to enhance zootechnical performances of peripartum' sows, possibly due to the modulation of the microbiota transmitted to the piglets.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry.

BACKGROUND: Hepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host. AIM: To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts. METHODS: Full-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay. RESULTS: NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC50) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98. CONCLUSION: Fused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32 µmol/L.

Citrus Extract Improves the Absorption and Utilization of Nitrogen and Gut Health of Piglets.

The purpose of this study was to investigate the effects of citrus extract (CE) on plasma free amino acids, intestinal morphology and enzymes activity, fecal nitrogen and phosphorus emissions in piglets. The experiment was performed on 144 weaned piglets (Duroc × Landrace × Large White) divided into three groups. Control (CON), fed a basic diet; Antibiotic (ANTI), fed a basic diet supplemented with 75 g/t chlortetracycline; Citrus extract (CE), fed a basic diet supplemented with 300 mL/t CE. The albumin content of the CE group was significantly higher than the CON group. Compared with the CON and ANTI groups, the CE group had increased concentrations of plasma total essential amino acids and threonine. Compared with the CON group, CE increased the α-aminoadipic acid concentration, while compared with ANTI group, it increased the 3-methylhistidine concentration. Compared with the CON group, the crypt depth of duodenum, jejunum and ileum decreased, and the ratio of villus height to crypt depth of ileum increased in the ANTI and CE groups. CE increased the activity of alkaline phosphatase and lipase in duodenum, and the activity of alkaline phosphatase and trypsin in jejunum. In brief, CE improved the absorption and utilization of nitrogen, intestinal morphology and digestive enzymes activity.

Antimicrobial efficacy of alternative compounds for use in oral care toward biofilms from caries-associated bacteria in vitro.

For caries-active patients, antimicrobial measures may be useful in addition to mechanical biofilm removal. The aim of this study was to investigate the antimicrobial efficacy of alternative compounds for use in oral care from two main categories (i.e., preservatives and natural compounds) toward biofilms from caries-associated bacteria as compared to oral care gold-standards chlorhexidine digluconate (CHX), cetylpyridinium chloride (CPC), and zinc. Compounds were screened in initial Streptococcus mutans biofilms. Then, the most effective compounds were further investigated in mature S. mutans and polymicrobial biofilms comprising Actinomyces naeslundii, Actinomyces odontolyticus, and S. mutans. Here, distinct treatment periods and concentrations were evaluated. Biofilms were visualized by scanning electron microscopy and bacterial membrane damage was evaluated by means of flow cytometry and staining with SYBR Green and propidium iodide. Citrus extract was the only compound exhibiting similar antimicrobial efficacy in initial S. mutans biofilms (>5 log10 ) as compared to CHX and CPC, but its effect was clearly inferior in mature S. mutans and polymicrobial biofilms. Flow cytometric data suggested that the mechanism of antimicrobial action of citrus extract may be based on damage of bacterial membranes similar to CHX and CPC. From all alternative compounds investigated in this study, citrus extract exhibited the highest antimicrobial efficacy toward in vitro biofilms from caries-associated bacteria, but still was less effective than oral care gold-standard antiseptics CHX and CPC. Nevertheless, citrus extract may be a valuable antimicrobial compound for use in oral care for caries-active patients.

Dietary Supplementation With Citrus Extract Altered the Intestinal Microbiota and Microbial Metabolite Profiles and Enhanced the Mucosal Immune Homeostasis in Yellow-Feathered Broilers.

The present study aimed to investigate the effects of citrus extract (CE) on intestinal microbiota, microbial metabolite profiles, and the mucosal immune status in broilers. A total of 540 one-day-old yellow-feathered broilers were randomly allotted into three groups and fed a basal diet (control group), or a basal diet containing 10 mg/kg of zinc bacitracin (antibiotic group), or 10 mg/kg of CE (CE group). Each treatment consisted of six replicates, with 30 broilers per replicate. After 63-day feeding, two broilers per replicate were randomly selected and slaughtered, and their ileal and cecal digesta and ileal tissue were collected for microbial composition, microbial metabolites, and gene expression analysis. The results showed that CE significantly increased the abundance of Barnesiella and Blautia than did the antibiotic group (adjusted P < 0.05), whereas it decreased the abundance of Alistipes and Bacteroides (adjusted P < 0.05). Meanwhile, the CE group also increased the numbers of Bifidobacterium and Lactobacillus than did the control and antibiotic groups (P < 0.05), whereas it decreased the number of Escherichia coli (P < 0.05). For microbial metabolites, dietary supplementation with CE increased the concentrations of lactate, total short-chain fatty acids, acetate, and butyrate in the cecum than did the control and antibiotic groups (P < 0.05), whereas it decreased the concentrations of amino acid fermentation products (ammonia, amines, p-cresol, and indole) (P < 0.05). Additionally, supplementation with CE up-regulated (P < 0.05) the mRNA expression of intestinal barrier genes (ZO-1 and Claudin) in the ileum than did both the control and antibiotic groups. However, antibiotic treatment induced gut microbiota dysbiosis, altered the microbial metabolism, and disturbed the innate immune homeostasis. In summary, these results provide evidence that dietary supplementation with CE can improve the intestinal barrier function by changing microbial composition and metabolites, likely toward a host-friendly gut environment. This suggests that CE may possibly act as an efficient antibiotic alternative for yellow-feathered broiler production.

Citrus limon Extract Loaded in Vesicular Systems for the Protection of Oral Cavity.

Background: The nanoincorporation of the extract of Citrus limon (L.) Osbeck var. pompia into liposomes was aimed at improving its antioxidant and antibacterial effects. Methods: The extract of the rind of Citrus limon (L.) Osbeck var. pompia was obtained by maceration in ethanol, evaporation, and freeze-drying. The extract phytochemical fingerprint was obtained by HPLC and mass spectrometry, and it was determined that gallic acid, neohesperidin, eriocitrin, and neoeriocitrin were the most abundant components. The freeze-dried extract was loaded in liposomes, glycerosomes, and penetration-enhancer-containing vesicles prepared with propylene glycol (PG-PEVs). Results: Capability of the vesicles of improving efficacy of the extract in counteracting oxidative stress was studied in vitro in keratinocytes, along with antimicrobial activity against planktonic cultures of Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis. Conclusion: Results showed that the vesicles, especially glycerosomes and PG-PEVs, prevented oxidative damage and cell death, and inhibited bacterial proliferation.

Antioxidant activity and influence of Citrus byproduct extracts on adherence and invasion of Campylobacter jejuni and on the relative expression of cadF and ciaB.

Adherence and invasion to cells are the key processes during infection development by Campylobacter jejuni (C. jejuni). In this study, extracts from the byproducts of Citrus limon, Citrus aurantium, and Citrus medica were added to the cultures of C. jejuni, and the adherence and invasion of C. jejuni to HeLa cells and the expression of cadF and ciaB genes were analyzed. The relative expression of the genes was determined by quantitative reverse transcription PCR (qRT-PCR). The antioxidant activity was determined using spectrophotometric methods. Byproduct extracts at subinhibitory concentrations affected the adherence (reduced 2.3 to 99%) and invasion (reduced 71.3 to 99.2%) to the HeLa cells. The expression of cadF and ciaB was reduced from 66 to 99% and from 81 to 99%, respectively. The total phenolic content of the byproducts varied from 92 to 26 mg GAE/g and the total flavonoids varied from 161 to 29.29 mg QE/g. C. aurantium showed the highest percentage of radical scavenging activity (RSA, 90.1). These extracts can prove as effective alternatives for devising new strategies to control Campylobacter infections.