Food supplement comprising neohesperidin dihydrochalcone and peppermint oil
By adding a combination of neohesperidin dihydrochalone and peppermint oil to animal feed, the problem of asymptomatic carrier transmission of Campylobacteria in poultry and pigs was solved, significantly reducing the carrying amount of Campylobacteria, and thus reducing the Human infection risk.
Patent Information
- Application Number
- CN202380048088.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-07-07
- Filing Date
- 2023-06-21
- Publication Date
- 2025-02-14
AI Technical Summary
Campylobacter disease is a zoonotic disease that is mainly transmitted through the consumption of contaminated chicken. The prior art is difficult to effectively reduce the transmission risk of asymptomatic carriers.
A food supplement containing neohesperidin dihydrochalone (NHDC) and peppermint oil (PMO) was used to add it to animal feed to reduce Campylobacter populations in poultry or pigs.
By adding a combination of NHDC and PMO to the diet of broiler or piglets, the carrying of Campylobacter in their bodies is significantly reduced, potentially reducing the occurrence of Campylobacteria in humans and limiting the use of antibiotics.
Smart Images
Figure CN119451587A_ABST
Abstract
Description
[0001] The present invention relates to a food supplement comprising neohesperidin dihydrochalcone and peppermint oil.
[0002] The invention also covers feed containing the food supplement and its use in reducing Campylobacter spp. in poultry or pigs, and proposes a method for reducing the risk of human infection with Campylobacter spp. Background Art
[0003] Campylobacteriosis is a worrisome zoonosis that poses a major human public health challenge in Europe, the United States, Australia and New Zealand. The disease is primarily transmitted through food, especially when chicken is contaminated with Campylobacter enterica serovars, which can be transmitted to humans through consumption and handling of contaminated meat. The main Campylobacter species are Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), which cause clinical symptoms such as diarrhea, abdominal pain, headache, nausea, vomiting, and in the worst cases, arthritis, irritable bowel syndrome (IBS) and Guillain-Barré syndrome (GBS). Therefore, asymptomatic carriage of Campylobacter in poultry is a major problem. Campylobacter jejuni colonizes the digestive tract of broiler chickens but is not usually pathogenic to the host under normal circumstances.
[0004] The use of antibiotics is the main way to reduce asymptomatic carriage of this bacterium, and the industry is looking for alternatives.
[0005] On the other hand, some plant-based compounds have shown positive effects on Campylobacter jejuni in vitro, mainly by reducing cell invasion or reducing the risk of transmission from asymptomatic carriers. Van Alphen et al. in 2012 [1] observed that at a dose of 0.25 mM, carvacrol was able to inhibit the motility of Campylobacter jejuni, while at a dose of 0.2 mM, it was able to effectively reduce the invasiveness of this strain to host cells.
[0006] Others have also studied the positive effects of curcumin against Campylobacter jejuni. For example, Lobo de Sa et al. proposed in 2019 [2] that curcumin can alleviate immune-mediated epithelial dysfunction caused by Campylobacter jejuni when co-cultured with immune cells in HT-29 cells at a dose of 50 μM.
[0007] Kovacs et al. (2019) conducted a detailed study on the effect of peppermint oil on Campylobacter jejuni NCTC 11168 cells [3]. They selected two sublethal concentrations of 50 μg / ml and 150 μg / ml to treat the cells and observed that peppermint oil at these concentrations could regulate the stress response and virulence characteristics of the bacteria. Under these experimental conditions, the minimum inhibitory concentration (MIC) was 100 μg / ml, and the minimum bactericidal concentration (MBC) was 400 μg / ml.
[0008] However, even though these products have shown some positive effects in in vitro testing, their effects on asymptomatic carriage in vivo have not yet been demonstrated. Summary of the invention
[0009] The main object of the present invention is to propose an animal food product which can be used as a preventive measure to reduce the number of Campylobacter jejuni in a host such as poultry or pigs.
[0010] The present invention more particularly relates to a food supplement according to clause 1 below:
[0011] 1. A food supplement comprising neohesperidin dihydrochalcone (NHDC) and peppermint oil
[0012] (PMO), preferably mixed.
[0013] Other advantageous features of the food supplements defined in clause 1 above are detailed in clauses 2 to 6 below:
[0014] 2. The food supplement according to clause 1, wherein the weight ratio of NHDC / PMO is in the range of 0.5 to 1.
[0015] 3. A food supplement according to clause 2, wherein the weight ratio of NHDC / PMO is in the range of 0.65 to 0.85.
[0016] 4. A food supplement according to clause 3, wherein the weight ratio of NHDC / PMO is 0.75.
[0017] 5. A food supplement according to any one of clauses 1 to 4, wherein NHDC and PMO are in solid form.
[0018] 6. A food supplement according to any one of clauses 1 to 4, wherein NHDC and PMO are in liquid form.
[0019] The present invention also relates to a feed according to the following clause 7:
[0020] 7. A feed comprising the food supplement according to items 1 to 6 above.
[0021] Other advantageous characteristics of the feeds defined in clause 7 above are detailed in clauses 8 to 10 below:
[0022] 8. Feed according to clause 7, comprising 10 ppm to 25 ppm of a food supplement according to clauses 1 to 6.
[0023] 9. Feed according to clause 8, comprising 15 ppm to 20 ppm of a food supplement according to clauses 1 to 6.
[0024] 10. Feed according to clause 9, comprising 17.5 ppm of the food supplement according to clauses 1 to 6.
[0025] According to another aspect, the present invention relates to the use as defined in clause 11 below:
[0026] 11. A food supplement as defined in clauses 1 to 6 or a feed as defined in clauses 7 to 10 for use in reducing
[0027] The load of Campylobacter spp.
[0028] According to another aspect, the present invention relates to the use as defined in clause 12 below:
[0029] 12. A food supplement according to clauses 1 to 6 or a feed according to clauses 7 to 10 for reducing
[0030] The load of Campylobacter spp.
[0031] According to another aspect, the present invention also relates to a method as defined in clause 13 below:
[0032] 13. A method of reducing the risk of Campylobacter infection in humans, comprising consuming poultry fed with a food supplement as defined in clauses 1 to 6 or a feed as defined in clauses 7 to 10.
[0033] According to another aspect, the present invention also relates to a method as defined in clause 14 below:
[0034] 14. A method of reducing the risk of Campylobacter infection in humans, comprising consuming pork fed with a food supplement as described in clauses 1 to 6 or a feed as described in clauses 7 to 10.
[0035] According to yet another aspect, the present invention relates to a composition comprising neohesperidin dihydrochalcone (NHDC) and peppermint oil (PMO) for use in reducing the load of Campylobacter in poultry and / or pigs.
[0036] According to yet another aspect, the present invention relates to a method for reducing the Campylobacter load in poultry and / or pigs by feeding the poultry and / or pigs with a composition comprising neohesperidin dihydrochalcone (NHDC) and peppermint oil (PMO).
[0037] Therefore, the inclusion of peppermint oil and NHDC in the diet of broiler chickens or piglets could reduce their Campylobacter carriage and thus potentially reduce the occurrence of campylobacteriosis in humans. Therefore, the use of antibiotics in poultry or pigs could be limited, thereby reducing the development of antibiotic resistance.
[0038] Other features and advantages of the present invention will now be described in detail in the following description, which refers to the accompanying drawings, which show:
[0039] [ Figure 1 ]: Schematic diagram of the in vitro experimental design;
[0040] [ Figure 2 ]: in vitro attachment results (combined results of two cell lines);
[0041] [ Figure 3 ]: Timeline representation of in vivo study design; and [ Figure 4 ]: Campylobacter bacterial counts on day 14 after cecal infection, expressed as log CFU / ml in the left panel or as log CFU / organ in the right panel. DETAILED DESCRIPTION
[0042] The inventors first selected different compounds to be included in the feed as preventive agents.
[0043] In the first step, an in vitro screening was performed to observe the adhesion ability of Campylobacter jejuni to two cell lines after pretreatment with different compounds.
[0044] In the second step, the two best products from the in vitro tests were selected and tested in a broiler challenge test to observe asymptomatic C. jejuni carriage levels in the cecum, which is known to represent carcass contamination.
[0045] Through the above tests, first in vitro and then in vivo, the inventors have observed a significant 1 log reduction in Campylobacter jejuni in broiler chickens at the end of the production cycle of broiler chickens or piglets using a combination of neohesperidin dihydrochalcone (NHDC) and peppermint oil (PMO).
[0046] According to a preferred embodiment, the weight ratio of NHDC / PMO in the food supplement is in the range of 0.5 to 1, more preferably in the range of 0.65 to 0.85. Ideally, the weight ratio of NHDC / PMO is about 0.75. NHCH and PMO are preferably uniformly mixed together.
[0047] Since NHDC is a solid raw material and PMO is a typical liquid raw material, additional product formulation is required to obtain a good feed inclusion. Therefore, either PMO is formulated as a solid granule or NHDC is dissolved to be able to combine the two raw materials in a closer physical state to obtain a homogeneous food supplement.
[0048] Different formulations can be produced. To convert the PMO into particles, in a first option, the PMO can be adsorbed / absorbed onto a carrier, which can be selected from any porous carrier, which can be selected from silica, sepiolite, diatomaceous earth, vermiculite, zeolite, porous starch. In a second option, the PMO can be treated to produce particles by introducing it into a liquid matrix, which can be solidified by using atomization or coagulation. The liquid matrix can include modified starch, cellulose derivatives, maltodextrins, gums, natural gums, waxes combined with amphiphilic molecules, fats or hydrogenated fats to be spray dried, spray granulated or spray cooled.
[0049] To convert NHDC into a liquid product, the product may be dissolved, solvated or emulsified in water or a solvent to be compatible with the PMO.
[0050] If the PMO is to be converted to a solid state, the product obtained can be mixed with NHDC directly or with the addition of a diluent. Another possibility is that spray agglomeration may be used in the process to agglomerate the NHDC onto the PMO particles.
[0051] The food supplement can be added to the feed of the animal, preferably poultry or pig. In this case, the feed can be added with 10ppm to 25ppm of the food supplement, and 15ppm to 20ppm of the food supplement is better. Ideally, the feed contains about 17.5ppm of the food supplement.
[0052] experiment
[0053] Two sets of tests have been performed:
[0054] - In vitro data looking at the attachment / penetration of C. jejuni in 2 cell lines, and
[0055] -In vivo challenge test in broiler chickens.
[0056] a) In vitro screening tests
[0057] In vitro challenge was performed with a new strain of Campylobacter jejuni MDR1 (showing multidrug resistance). The bacteria were cultured in microaerobic jars.
[0058] The following media were used for testing:
[0059] William's E medium (BioWhittaker reference number BE12-761F)
[0060] Dulbecco's Modified Eagle Medium (DMEM) 4.5 g glucose / L (Gibco reference number 41965-039)
[0061] Fetal bovine serum (FBS) (Sigma reference number F7524)
[0062] L-Glutamine 200 mM (Gibco Ref. 25030-024)
[0063] Gentamycin 50 mg / mL (Gibco reference number 1570037)
[0064] PBS (Sigma reference number D8662)
[0065] Ethanol 96° (Carlo-Erba reference 528151)
[0066] Blood agar
[0067] In vitro testing was performed using 2 cell lines: HT-29 , which is a human colorectal adenocarcinoma cell line with epithelial morphology; and LHM , which is a Gallus gallus hepathocarcinoma cell line with epithelial morphology.
[0068] The two cell lines were cultured in the following culture media: LHM was cultured in 10% fetal bovine serum (FBS) or FBS and William's E medium, while HT-29 was cultured in a medium containing 1% glutamine, 10% FBS and DMEM 4.5 g glucose / L.
[0069] Table 1: List of tested products, their combinations and associated dosages
[0070]
[0071] *The peppermint oil used is peppermint essential oil or peppermint extract, the main components of which are shown in Table 2
[0072] Table 2: Specifications of peppermint oil or extract
[0073] molecular Minimum weight% Maximum weight % Eucalyptus 2.5 7 Menthone 10 30 Menthol 20 40 Menthone 0.0 4 L-Menthyl acetate 0.5 8 Germacene D 0.2 2 Menthofuran 3 7
[0074] The effects of compounds were compared to controls based on 10 μl 96% ethanol. All tests were performed in microwells.
[0075] The in vitro challenge of the two cell lines was as follows:
[0076] · The C. jejuni load was set at 8 log CFU, which was the dose used for in vivo experiments.
[0077] The multiplicity of infection (MOI) is defined as the ratio between the number of bacteria and the number of potential host cells, or the number of bacteria used for infection (CFU) / number of host cells (UFC) is set to 100.
[0078] The contact time is set at 30 minutes.
[0079] Infect confluent cells with bacterial suspension.
[0080] Six replicates were performed for each treatment and cell line, with at least one replicate per day if testing was done on different days.
[0081] The main time setting scheme for in vitro experimental design is as follows Figure 1 The plate counts of Campylobacter jejuni were then analyzed and the average log CFU / well of the two cell lines combined is shown in Figure 2 .
[0082] From the in vitro tests, it was observed that each product alone did not significantly reduce the attachment and penetration of C. jejuni into cells, but the combination of mint and NHDC significantly reduced the attachment or penetration of C. jejuni into cells, highlighting the synergistic effect of using the product combination.
[0083] b) In vivo testing
[0084] The combination obtained above was further tested in vivo. The description of the in vivo challenge test is introduced below.
[0085] In Table 3 the tested products and their concentrations in the feed are disclosed.
[0086] Table 3: Target content of active substances or plant extracts in feed
[0087]
[0088] **Same as above
[0089] The experiment lasted 35 days, during which time the chickens' growth was tracked.
[0090] From day 0 to day 35, broiler chickens were fed dietary treatments via feed.
[0091] Bacterial inoculation was performed on day 21. Necropsies and cecal analyses were performed on days 4, 10 and 14 post-inoculation (corresponding to days 25, 31 and 35 of age) to follow cecal colonization as a function of treatment (7 / treatment).
[0092] The following experimental design was applied:
[0093] Pre-test setup
[0094] (i) Collect eggs from the hatter,
[0095] (ii) Decontamination and incubation of eggs within 21 days, experimental setup
[0096] (i) One-day-old chicks arrive in straw pens,
[0097] (ii) J-7 (before challenge): 3 broilers / treatment group were used to check the health status of chicks (Campylobacter, Salmonella, Clostridium perfringens, Enteropathogenic Escherichia coli).
[0098] (iii) Broiler chickens were reared to 21 days of age (animal technical follow-up, collection and weighing of feed rejections and collection of weight gain data),
[0099] (iv) On day 21, 2.07 x 10 8 jejuni CJ-MDR1 were inoculated with 0.2 ml of cfu and followed up for carriage until the end of the protocol (animal technical follow-up, weight gain, feed intake with unsupplemented and supplemented feed),
[0100] (v) Necropsies were performed on days 4, 10 and 14 post infection (pi), with 7 broilers per treatment. At each necropsy, 2 cecums + tonsils were sampled for bacterial numbering (analyzed by Inovalys).
[0101] feed
[0102] Animals were treated with the product PIP-A: NHDC and peppermint oil were incorporated into the feed throughout the protocol. The diet was formulated into pellets of 6 mm length x 2.5 mm diameter. The composition of the diet ingredients is shown in Table 4.
[0103] Table 4: Composition of the control diet
[0104]
[0105]
[0106] *Anticoccidial drug SACOX
[0107] The birds were fed a monophasic diet throughout the trial. Product PIP-A was added to the above basal diet in a diluted form, contained within the range of 0.166% of the diet, which was not expected to affect the overall diet composition, in order to provide 7.5 ppm NHDC and 10 ppm peppermint oil in the supplementary feed.
[0108] exist Figure 3 The overall in vivo experimental design is schematically presented in .
[0109] exist Figure 4 The results of the in vivo tests are presented in , in particular the bacterial counts of Enterobacter campylobacter at the end of the production cycle (day 35 corresponding to day 14 post infection).
[0110] The results showed that the combination of peppermint oil and NHDC helped to significantly reduce the Campylobacter jejuni carriage in the cecum of chickens 14 days after infection, with a reduction of about 1 log, and the effect was very significant (P<0.007).
[0111] Examples of NHDC / PMO food supplement formulations
[0112] Example 1: Powder Formulation 1
[0113] raw material weight% NHDC 0.8 PMO 1.3 Hydrogenated fats 11.7 Thinner 86.2 total 100
[0114] Example 2: Powder Formulation 2
[0115] raw material weight% NHDC 0.5 PMO 0.6 Hydrogenated fats 5.4 Silicon dioxide 1.4 Calcium carbonate 10.0 Second powder 82.1 total 100
[0116] Example 3: Powder Formulation 3
[0117] raw material weight% NHDC 0.5 PMO 0.6 Silicon dioxide 98.9 total 100
[0118] Example 4: Powder Formulation 4
[0119] raw material weight% NHDC 0.8 PMO 1.3 Maltodextrin 87.9 gum 10.0 total 100
[0120] Example 5: Liquid Formulation 1
[0121] raw material weight% NHDC 0.5 PMO 0.6 Glyceryl Polyglycol Ricinoleate 5.4 preservative 0.5 water 93.0 total 100
Claims
1. A food supplement comprising neohesperidin dihydrochalcone (NHDC) and peppermint oil (PMO).
2. The food supplement according to claim 1, wherein the weight ratio of NHDC / PMO is in the range of 0.5 to 1.
3. The food supplement according to claim 2, wherein the weight ratio of NHDC / PMO is in the range of 0.65 to 0.
85.
4. The food supplement according to claim 3, wherein the weight ratio of NHDC / PMO is 0.
75.
5. The food supplement according to any one of claims 1 to 4, wherein NHDC and PMO are in solid form.
6. The food supplement according to any one of claims 1 to 4, wherein NHDC and PMO are in liquid form.
7. A feed comprising the food supplement according to claims 1 to 6.
8. A feed according to claim 7, comprising 10 ppm to 25 ppm of a food supplement according to claims 1 to 6.
9. A feed according to claim 8 comprising 15 ppm to 20 ppm of a food supplement according to claims 1 to 6.
10. Feed according to claim 9, comprising 17.5 ppm of a food supplement according to claims 1 to 6.
11. A food supplement according to claims 1 to 6 or a feed according to claims 7 to 10 for use in reducing the Campylobacter load in poultry.
12. A food supplement according to claims 1 to 6 or a feed according to claims 7 to 10 for use in reducing the Campylobacter load in pigs.
13. A method of reducing the risk of infection of humans with Campylobacter, comprising consuming poultry meat fed with a food supplement according to claims 1 to 6 or a feed according to claims 7 to 10.
14. A method of reducing the risk of Campylobacter infection in humans, comprising consuming pork fed with a food supplement as claimed in claims 1 to 6 or a feed as claimed in claims 7 to 10.