The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

BACKGROUND: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. RESULTS: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500 g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon's diversity and Pielou's Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q < 0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2 and 1% in the 300 and 500 g/MT. These changes in the microbial consortia were statistically (Q < 0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500 g/MT compared to NTC and 300 g/MT (P < 0.05). CONCLUSION: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

Reduction of nitrogen excretion and emission in poultry: A review for organic poultry.

Organic poultry is an alternative to conventional poultry which is rapidly developing as a response to customers' demand for better food and a cleaner environment. Although organic poultry manure can partially be utilized by organic horticultural producers, litter accumulation as well as excessive nitrogen still remains a challenge to maintain environment pureness, animal, and human health. Compared to conventional poultry, diet formulation without nitrogen overloading in organic poultry is even more complicated due to specific standards and regulations which limit the application of some supplements and imposes specific criteria to the ingredients in use. This is especially valid for methionine provision which supplementation as a crystalline form is only temporarily allowed. This review is focused on the utilization of various protein sources in the preparation of a diet composed of 100% organic ingredients which meet the avian physiology need for methionine, while avoiding protein overload. The potential to use unconventional protein sources such as invertebrates and microbial proteins to achieve optimal amino acid provision is also discussed.

Characterization of isolated yeast growth response to methionine analogs.

Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.

Application of orange essential oil as an antistaphylococcal agent in a dressing model.

BACKGROUND: Staphylococcus aureus is the pathogen most often and prevalently involved in skin and soft tissue infections. In recent decades outbreaks of methicillin-resistant S. aureus (MRSA) have created major problems for skin therapy, and burn and wound care units. Topical antimicrobials are most important component of wound infection therapy. Alternative therapies are being sought for treatment of MRSA and one area of interest is the use of essential oils. With the increasing interest in the use and application of natural products, we screened the potential application of terpeneless cold pressed Valencia orange oil (CPV) for topical therapy against MRSA using an in vitro dressing model and skin keratinocyte cell culture model. METHODS: The inhibitory effect of CPV was determined by disc diffusion vapor assay for MRSA and vancomycin intermediate-resistant S. aureus (VISA) strains. Antistaphylococcal effect of CPV in an in vitro dressing model was tested on S. aureus inoculated tryptic soya agar plate. Bactericidal effect of CPV on MRSA and VISA infected keratinocyte cells was examined by enumeration of extra- and intra-cellular bacterial cells at different treatment time points. Cytotoxic effects on human skin cells was tested by adding CPV to the keratinocyte (HEK001) cells grown in serum free KSFM media, and observed by phase-contrast microscope. RESULTS: CPV vapour effectively inhibited the MRSA and VISA strains in both disc diffusion vapour assay and in vitro dressing model. Compared to untreated control addition of 0.1% CPV to MRSA infected keratinocyte decreased the viable MRSA cells by 2 log CFU/mL in 1 h and in VISA strain 3 log CFU/mL reduction was observed in 1 h. After 3 h viable S. aureus cells were not detected in the 0.2% CPV treatment. Bactericidal concentration of CPV did not show any cytotoxic effect on the human skin keratinocyte cells in vitro. CONCLUSIONS: At lower concentration addition of CPV to keratinocytes infected with MRSA and VISA rapidly killed the bacterial cells without causing any toxic effect to the keratinocytes. Therefore, the results of this study warrant further in vivo study to evaluate the potential of CPV as a topical antistaphylococcal agent.

Electrostatic spraying of food-grade organic and inorganic acids and plant extracts to decontaminate Escherichia coli O157:H7 on spinach and iceberg lettuce.

UNLABELLED: The prevalence of foodborne illnesses is continually on rise. In the U.S.A., Escherichia coli O157:H7 (E. coli) has been associated with several outbreaks in minimally processed foods. Spinach and lettuce pose higher food safety risks and recurring food recalls suggest the insufficiency of current disinfection strategies. We aimed at offering a natural antimicrobial alternative using organic acids (malic, tartaric, and lactic acids [MA, TA, and LA, respectively]) and grape seed extract (GSE) and a novel application method using electrostatic spraying to evenly distribute the antimicrobials onto produce. Spinach and lettuce samples were washed, sanitized with sodium hypochlorite solution (6.25 mL/L), dip inoculated in water containing E. coli (7.0 log CFU/mL) for 24 h, and rewashed with sterile water to remove nonadhered pathogens. The samples were sprayed electrostatically with MA, LA, and GSE alone and in combinations and for comparison, with phosphoric acid (PA) and pH controls with deionized water adjusted to 1.5/2.3/3.6 and stored at 4 °C. When combined with LA (3%), MA (3%) showed 2.1 to 4.0 log CFU/g reduction of E. coli between the days 1 and 14 on spinach and 1.1 to 2.5 log CFU/g reduction on lettuce. Treatment with PA (1.5%) and PA (1.5%)-GSE (2%) exhibited 1.1 to 2.1 log CFU/g inhibition of E. coli on spinach during the 14-d storage. Our findings demonstrated the efficacy of electrostatic spraying of MA, LA, and GSE on fresh produce to improve the safety and lower the public health burden linked to produce contamination. PRACTICAL APPLICATION: Electrostatic spraying is an emerging technique that can be adopted to improve the distribution and application of antimicrobials during fresh produce sanitation. Relatively simple and quick, the process can access most/all parts of produce surface and offer protection from food pathogens. The use of malic and lactic acids with or without grape seed extract can serve as effective antimicrobials when sprayed electrostatically, lowering the risk from postcontamination issues with spinach and iceberg lettuce. This application technology can be extended to improve the commercial food safety of other produce, fruits, poultry, and meat.

Inhibition of beef isolates of E. coli O157:H7 by orange oil at various temperatures.

UNLABELLED: Plant essential oils have previously been shown to exhibit antimicrobial activities against various microorganisms. In this study, cold pressed terpeneless Valencia orange oil (CPTVO) was examined at various temperatures (37, 10, and 4 °C) to determine its antimicrobial activity against 3 strains of E. coli O157:H7 recovered from beef products. A micro broth dilution method using 96 well microtiter plates was used with trypticase soy broth with 0.15% agar and 2,3,5 tetrazolium chloride as a growth indicator as the medium. Serial dilutions of CPTVO were made, resulting in final concentrations of oil ranging from 0.2% to 25% or 0.1% to 10%. Plates were incubated statically at 4, 10, or 37 °C, and sampled hourly. After 6 h at 37 °C, all strains were inhibited at concentrations ranging from 0.2% to 0.6%, with a mean of 0.4 ± 0.01%. At 10 °C, all strains were inhibited at concentrations ranging from 0.8% to 6.3%, with a mean of 1.1% ± 0.2%, after 6 h. At 4 °C, all strains were inhibited after 6 h at concentrations ranging from 2.3% to 4.6%, with a mean of 3.5% ± 2.1%. After 24 h at 4 °C the strains were inhibited at concentrations ranging from 0.7% to 1% with a mean of 0.8% ± 0.3%. The ranges appear to be the result of effects from the variable nature of a complex media and an antimicrobial that presents potential multiple mechanisms for inhibition. It appears CPTVO is a viable option to inhibit E. coli O157:H7 growth at refrigeration temperatures. PRACTICAL APPLICATION: Beef products are often the source of foodborne illness from the organism E. coli O157:H7. Orange essential oils have been in the human diet for centuries, and the research reported here indicates that some of these oils may be used as surface applications during cold temperatures to inhibit the foodborne pathogen E. coli O157:H7.

In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance.

We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. abscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.

Enhanced inhibition of Listeria monocytogenes by a combination of cold pressed terpeneless Valencia orange oil and antibiotics.

This study was designed to evaluate the ability of cold pressed terpeneless Valencia orange oil (CPTVO) to enhance the effectiveness of antibiotics against 10 strains of Listeria monocytogenes. Disc diffusion assays were performed to determine the effects of CPTVO and two antibiotics with different mechanisms of action (i.e., penicillin and chloramphenicol) individually and in combination with CPTVO. CPTVO alone produced zones ranging from 16.5 to 19.9 mm. Penicillin at 2 or 10 units produced zones ranging from <6 to 13.4 mm, and from 16 to 19.5 mm, respectively. Chloramphenicol at 5 or 30 µg had zones ranging from <6 to 6.9 mm, and from 10.8 to 15.9 mm, respectively. Penicillin (2 and 10 units) plus CPTVO produced zones ranging from 20.2 to 25.3 mm, and from 21.9 to 28 mm, respectively. Chloramphenicol (5 or 30 µg) plus CPTVO produced zones of from 20.1 to 26.6 mm, and from 19.5 to 23.9 mm, respectively. In conclusion, the combination of antibiotics with CPTVO increases their ability to inhibit L. monocytogenes.

Inhibition of Listeria monocytogenes by exposure to a combination of nisin and cold-pressed terpeneless Valencia oil.

Listeria monocytogenes is an important foodborne pathogen and its control in foods is a significant challenge. This study evaluated the effectiveness of nisin and cold-pressed terpeneless Valencia oil (CPTVO) on limiting L. monocytogenes growth. Disk diffusion assays were performed to determine the effects of CPTVO and nisin individually and in combination. Together, these antimicrobials produced a zone of inhibition that was significantly larger (P < 0.05) than zones correlating to CPTVO or nisin individually. Furthermore, L. monocytogenesΔsigB had an increased sensitivity to the combination treatment. Growth experiments performed in brain heart infusion (BHI) broth revealed the effects of nisin and CPTVO, individually and in combination on L. monocytogenes growth rate. When L. monocytogenes was grown in BHI containing 0.025% CPTVO and 26 IU/mL nisin, no growth inhibition was observed relative to the control. However, exposure to CPTVO at 0 h followed by the introduction of nisin at 15 h resulted in a statistically significant (P < 0.05) reduction in growth. This approach to inhibiting L. monocytogenes has potential as an all-natural, generally-recognized-as-safe multiple hurdle intervention that may be applicable for ready-to-eat products in which L. monocytogenes is likely to cause foodborne illness.

Activity of citrus essential oils against Escherichia coli O157:H7 and Salmonella spp. and effects on beef subprimal cuts under refrigeration.

UNLABELLED: Escherichia coli O157:H7 and Salmonella spp. are bacterial pathogens often associated with beef, and cause many cases of foodborne illness each year in the United States. During beef slaughter and processing, these bacteria may spread from the hide or intestines to the carcass. The objective of this research was to investigate the use of naturally occurring compounds citrus essential oils (CEOs) extracted from orange peel to reduce or eliminate these pathogens at the chilling stage of processing, or during fabrication. Brisket flats (used to simulate beef subprimals) were spot inoculated with approximately 6 log of surrogate generic E. coli cocktail (previously shown to be identical in growth and survival parameters to E. coli O157:H7 and Salmonella spp.). Following drying, CEOs were applied by spraying at concentrations of 3% and 6% to the surface of different pieces of meat. Treatments were applied using a custom built spray cabinet at 2.07 bar and applied at a rate of 3.79 L/min to replicate commercial practices. The CEOs significantly reduced (P < 0.05) the concentration of E. coli on the brisket flats in comparison to inoculated no spray or water sprayed controls over a period of 90 d, while causing an initial reduction of approximately 1.4 log units. Total aerobic bacteria and psychrotrophic counts were also reduced on uninoculated briskets following treatment. These results indicate that 3% cold-pressed terpeneless Valencia orange oil could be used as an additional intervention against E. coli O157:H7 and Salmonella spp. at the refrigerated storage stage of processing. PRACTICAL APPLICATION: CEOs are natural compounds that have been designated as Generally Recognized as Safe (GRAS). They can be used to control Salmonella spp. and E. coli O157:H7 on beef carcasses at the chilling stage.

Effect of soluble maillard reaction products on cadA expression in Salmonella typhimurium.

The presence of Maillard reaction products (MRP) in foods and food components is due to the non-enzymatic reaction between protein and carbohydrate residues triggered by thermal steps during food processing. The objective of this study was to assess the effect of MRPs and increasing lysine concentrations on S. Typhimurium growth and the expression of cadA which may be an indirect determinant of Salmonella virulence response. Variations in lysine concentrations (from 0 to 0.5 mM) did not exert any effect either on the final optical density after 6-hour incubation or the growth rates of S. Typhimurium in media containing MRPs. In contrast to the reduced final absorbancy of the bacterial cultures grown with histidine and arginine MRPs supplementations (0.1%), growth rates, in general, remained unaltered by all MRPs at each lysine concentration when compared to the control (M9 pH 5.8, no MRPs added). The induction levels of cadA in media containing 0.1% MRPs were close to cadA induction in the reference media (M9, pH 5.8 and no MRPs) and did not exceed the corresponding values by more than approximately 30%. Although the observed negligible induction of cadA under these conditions complies with the concept of its potential "anti-virulence" function, additional studies involving various concentrations and more refined MRPs are needed.

Microbial inhibitory and radical scavenging activities of cold-pressed terpeneless Valencia orange (Citrus sinensis) oil in different dispersing agents.

BACKGROUND: Due to their low solubility in water, oil-based bioactive compounds require dispersion in a surface-active agent or appropriate solvents to ensure maximum contact with microorganisms. These combinations, however, may change their physical and/or chemical characteristics and consequently alter the desired functionality. The objective of this study was to determine the impact of selected dispersing agents, ethanol, dimethyl sulfoxide (DMSO), and Tween-80, on cold-pressed terpeneless (CPT) Valencia orange oil to function as a free radical scavenger and an antimicrobial food additive. RESULTS: When dissolved in ethanol or DMSO, the orange oil fraction had similar minimum inhibitory concentrations (MIC) for Listeria monocytogenes ATCC 19 115 (0.3% and 0.25% v/v respectively), which were significantly lower (P

Electrostatic sprays of food-grade acids and plant extracts are more effective than conventional sprays in decontaminating Salmonella Typhimurium on spinach.

About 40000 people fall victim to Salmonella infections every year in the United States. Recent occurrences of Salmonella contaminated spinach and its recalls have accelerated the need for efficient antimicrobials targeting these pathogens. Our study was aimed at evaluating the inhibitory properties of malic, tartaric, and lactic acids, and grape seed extract (GSE) alone and in combinations and their application methods against Salmonella Typhimurium-inoculated spinach using a response surface method. Fresh spinach leaves were washed, disinfected with sodium hypochlorite solution (0.04% v/v), rewashed with sterile deionized (DI) water, and inoculated with a 2nd-day culture of S. Typhimurium (7.0 log CFU/mL). Adhered S. Typhimurium population on day 0 were 7.5 log CFU/g. These were treated with individual and combinations of organic acids with GSE or DI water (control) adjusted to the same pH as that of the test solutions with both the modes of application and leaves were refrigerated at 4 °C. Malic acid (2%) in combination with GSE (3%) or lactic acid (3%) sprayed electrostatically showed reductions of 2.6 to 3.3 log CFU/g compared to lower log reductions (0.0 to 0.3 log CFU/g) by day 14 if sprayed conventionally. These findings indicate that malic acid in combination with GSE/lactic acid solutions applied by electrostatic spraying exhibited higher inhibition of pathogens than conventional spraying and can be used for commercial applications to enhance food safety.

Campylobacter and Arcobacter species sensitivity to commercial orange oil fractions.

Seven orange oil fractions were screened for their ability to inhibit the growth of selected Campylobacter and Arcobacter spp. using the standard agar-disk diffusion assay. Cold pressed (CP) terpeneless Valencia orange oil was found to be the most inhibitory to both Campylobacter jejuni and Campylobacter coli, exhibiting maximum zones of inhibition up to 80+/-0.0 mm. Five-fold concentrated Valencia oil and distilled d-limonene resulted in Campylobacter inhibition zones ranging from 11.0+/-1.4 to 44+/-1.4 mm against both C. jejuni and C. coli. No inhibition of Arcobacter spp. was detected by 6 out of 7 orange fractions except CP terpeneless Valencia orange oil which produced inhibition zones varying from 9.5+/-0.7 to 29+/-1.4 mm. Naturally occurring C. jejuni UAF 244 was isolated from a whole retail chicken, confirmed by hippuricase gene PCR assay, and used to determine antimicrobial capacities of the CP terpeneless Valencia orange oil and limonene when applied on chicken legs and thighs. The two types of chicken parts did not influence the antimicrobial strength of both orange fractions. While the observed reduction of C. jejuni cells attached to the skin varied approximately 1.5 to 2 logarithms compared to the control, the growth inhibition of the bacterial cells by limonene in the rinse increased by 6-fold and complete inhibition without recovery of detectable viable cells occurred when CP Valencia orange oil was applied. The study demonstrated the potential of the selected commercial orange oil fractions to serve as natural antimicrobials against C. jejuni, C. coli, and Arcobacter spp.