A method for synergistically regulating the color of eggshells of poultry by plant essential oil-probiotics

Abstract

The application discloses a kind of plant essential oil-probiotics synergistic regulation method of eggshell color of poultry, belong to poultry breeding technical field, the method includes: preparation of microcapsule coated composite probiotic powder of inlaying L-hydroxyproline chelated copper, composite probiotic includes lactobacillus plantarum, bacillus subtilis, enterococcus faecalis and saccharomyces cerevisiae;Preparation of oil-in-water type compound emulsified plant essential oil preparation, compound essential oil includes oregano oil, rosemary oil, cinnamon oil and thyme oil;For egg production peak maintenance period, eggshell color correction period and acute stress emergency period, respectively using different feed or drinking water addition scheme is applied.This application is through the ternary synergistic effect of probiotic, plant essential oil and L-hydroxyproline chelated copper, combined with phased accurate application, can quickly deepen eggshell color, improve the uniformity of coloring and long-term prevent discoloration, in the effect of improving eggshell redness, reducing the proportion of light shell egg and shortening stress recovery time is remarkable.

Description

Technical Field

[0001] This invention mainly relates to the field of poultry farming technology, specifically a method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics. Background Technology

[0002] Eggshell color is an important indicator of the appearance quality of poultry eggs, directly affecting consumers' purchasing intentions and the commercial value. Brown-shelled laying hens often experience problems such as lighter eggshell color, increased color variation, and an increase in mottled eggs during the later stages of egg production or due to stress factors such as high temperatures, flock transfers, and immunization, causing significant economic losses to farmers. Therefore, developing technical methods to effectively control eggshell color has important practical value.

[0003] Currently, there are numerous research reports on using plant essential oils or probiotics to improve poultry egg quality. Regarding plant essential oils, studies have shown that adding them to feed can improve egg production performance and egg quality in laying hens. For example, adding oregano oil can enhance eggshell strength and yolk color, and can improve eggshell mechanical properties and ultrastructure by reducing uterine inflammation in laying hens. Research from the Feed Research Institute of the Chinese Academy of Agricultural Sciences has for the first time elucidated the biological mechanism of eggshell fading in the later stages of laying, discovering a close correlation between uterine microbial community structure and eggshell color, and subsequently screening for bacteria with regulatory potential. Regarding probiotics, literature reports that adding probiotic microecological preparations to feed can significantly improve albumen height, yolk color, and eggshell color in Lohmann Brown laying hens; compound microecological preparations have also been shown to improve eggshell strength and yolk color. Furthermore, research on the combined application of plant essential oils and probiotics has also been conducted. For example, studies on the effects of combined oregano oil and probiotics on the production performance, egg quality, and serum antioxidant levels of Lohmann Brown laying hens showed that the combination significantly affected the feed conversion ratio and egg production rate, but eggshell color was not the primary indicator, and the improvement was limited. Studies on compound preparations of Bacillus subtilis and plant essential oils also confirmed their effect on improving the yolk index, but similarly, eggshell color was not the core objective.

[0004] However, the aforementioned existing technologies still have the following shortcomings: First, while plant essential oils and probiotics can improve eggshell strength and yolk color when used alone, their specific regulatory effect on eggshell color is not significant or is not the focus of research. Most literature only observes eggshell color as a secondary indicator of egg quality, lacking targeted regulation schemes.

[0005] Second, although there have been reports on the combined application of plant essential oils and probiotics, their focus is mainly on growth performance, gut health and antioxidant capacity, and eggshell color has not yet been taken as a core technical target. Moreover, the combined use of the two has a limited effect on improving eggshell color, and no technical solution specifically for synergistic regulation of eggshell color has been found.

[0006] Third, existing technologies all employ a fixed-dose, continuous application method, failing to establish differentiated dosages and administration routes for different physiological stages of laying hens (such as the peak laying maintenance period, the late laying correction period, and the acute stress rescue period). This results in unsatisfactory effects in older laying hens or under stress, and also suffers from problems such as excessive dosage and slow onset of action. Furthermore, there are no research reports or patent disclosures in the existing technologies regarding the combined use of L-hydroxyproline chelated copper with plant essential oils and probiotics for eggshell color regulation.

[0007] Therefore, those skilled in the art urgently need to develop a method that can synergistically regulate multiple targets, apply precisely in stages, rapidly improve and maintain eggshell color for a long time, in order to overcome the shortcomings of existing technologies such as non-specific effects, poor adaptability and targeting, and low dosage efficiency. Summary of the Invention

[0008] This invention addresses the problem of overly simplistic solutions in existing technologies by providing a significantly different approach. It offers a method for the synergistic regulation of poultry eggshell color using plant essential oils and probiotics. This method solves the problems mentioned in the background section regarding the lack of multi-target synergistic regulation and precise, phased application methods for improving eggshell color. These problems result in insignificant eggshell color improvement, poor adaptability to older laying hens and those under stress, slow effectiveness, and low dosage efficiency.

[0009] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for synergistic regulation of poultry eggshell color using plant essential oils and probiotics. The core of this method lies in embedding L-hydroxyproline-chelated copper within a compound probiotic microcapsule, which is then used in conjunction with a compound emulsified plant essential oil. Differentiated dosages and administration routes are employed based on different physiological stages of the poultry. After the product enters the digestive tract, the microcapsules release the product slowly in the intestines. The probiotics colonize and break down L-hydroxyproline for energy, releasing Cu... 2+ Reversible complexation with the phenolic hydroxyl groups of essential oils achieves sustained-release antioxidant effects, while Cu 2+ It acts directly as a coenzyme in the synthesis of protoporphyrin IX. Through the synergistic effect of three pathways—probiotics improving the gut / uterine microenvironment, essential oils enhancing pigment synthase activity, and copper ions catalyzing pigment synthesis—it achieves the technical effects of rapidly deepening eggshell color, uniform coloring, and long-lasting prevention of fading. This method is applicable to laying hens, ducks, quails, and other egg-laying poultry.

[0010] Specifically, a method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics includes the following steps: S1. Preparation of microcapsule-coated compound probiotic powder containing L-hydroxyproline-chelated copper: (1) Preparation of compound probiotic bacterial solution: By mass percentage, take 30-40% Lactobacillus plantarum, 20-30% Bacillus subtilis, 15-25% Enterococcus faecalis, and 15-25% Saccharomyces cerevisiae. After activating each strain separately, inoculate Lactobacillus plantarum and Enterococcus faecalis into MRS liquid medium at a total inoculation amount of 5% (v / v), inoculate Bacillus subtilis into LB liquid medium, and inoculate Saccharomyces cerevisiae into YPD liquid medium. After incubation at 37℃ for 24 h, collect the bacterial cells by centrifugation, mix them, wash twice with sterile physiological saline, and resuspend to a bacterial concentration of 1×10⁻⁶. 10 The concentration of CFU / mL was used to obtain a compound bacterial solution; the total amount of Lactobacillus plantarum, Bacillus subtilis, Enterococcus faecalis, and Saccharomyces cerevisiae was 100%. (2) Adding L-hydroxyproline chelated copper: Weigh 15% to 20% (by mass) of L-hydroxyproline chelated copper powder into the compound bacterial solution, and slowly add it to the compound bacterial solution, stirring until dissolved. The "dry weight of the bacterial cells" is pre-calibrated by drying an equal volume of bacterial solution at 105°C to a constant weight. The preferred addition amount is 17% of the dry weight of the bacterial cells. This addition amount is calculated to make the copper content in the final probiotic powder with embedded chelated copper reach about 0.05% (500 mg / kg). Combined with the addition amount in the S3 stage, it can supplement the copper content in the feed to 80 to 150 mg / ton, which meets the national standards for the safe use of feed additives.

[0011] (3) Preparation of microcapsule coating wall material: Sodium alginate (viscosity 200-400 mPa·s) and maltodextrin (DE value 15-20) are mixed at a mass ratio of 1:2. Deionized water is added to prepare a wall material solution with a total concentration of 6% (w / v). The solution is then autoclaved at 121°C for 15 min and cooled to room temperature for later use.

[0012] (4) Preparation of microcapsules with embedded copper chelates: The above-mentioned composite bacterial solution with added L-hydroxyproline chelate copper was mixed with the wall material solution at a volume ratio of core material: wall material = 1:2, and stirred to disperse evenly. A low-temperature spray drying microencapsulation device (model B-290, BUCHI or equivalent) was used, with the inlet air temperature set at 160-170℃, the outlet air temperature at 80-85℃, the atomization pressure at 0.2MPa, the feeding speed at 30-50mL / min, and the nozzle diameter at 0.7mm. The dried powder was collected and passed through an 80-mesh sieve to obtain the microcapsule-coated composite probiotic powder with embedded L-hydroxyproline chelate copper (hereinafter referred to as copper-encapsulated probiotic powder). The total number of viable bacteria in the obtained product is ≥5×10 9CFU / g, gastric survival rate ≥85%, moisture content ≤8%, viable bacteria attenuation rate ≤5% / month after 12 months of sealed storage at room temperature.

[0013] S2. Preparation of emulsified plant essential oil formulations: (1) Blended essential oil stock solution: By volume percentage, take 45-55% oregano oil, 25-35% rosemary oil, 8-16% cinnamon oil, and 5-12% thyme oil, mix them at room temperature and stir evenly to obtain the blended essential oil stock solution; the total amount of oregano oil, rosemary oil, cinnamon oil, and thyme oil is 100%; (2) Preparation of the emulsion system: Based on the total mass of the final emulsion, take 75-85 parts (by weight) of deionized water, add 6-10 parts of food-grade soybean lecithin and 1-3 parts of Tween-80, and stir in a 40°C water bath until completely dissolved to form an aqueous phase; then, under high-speed shearing conditions (shearing speed of 8000-10000 r / min), slowly add 10 parts of the compound essential oil mother liquor (i.e., the mass ratio of aqueous phase to oil phase is 9:1), and continue shearing for 8-10 min after the addition is complete. After shearing, let stand at room temperature for 30 min to defoam, and the water-in-oil type compound emulsified plant essential oil preparation (referred to as emulsified plant essential oil preparation) is obtained. The preparation is a milky white homogeneous liquid. After being stored at 25°C in a sealed, light-proof container for 6 months, the effective active ingredient retention rate is ≥95%, with no stratification, no precipitation, and no volatilization failure.

[0014] S3. Precise formulation and application according to physiological stages: The probiotic powder with embedded chelated copper prepared in S1 was compounded with the emulsified plant essential oil preparation prepared in S2 according to different physiological stages. Before compounding, it was confirmed that the powder and emulsion mixed at room temperature without clumping or precipitation.

[0015] (1) Peak egg production maintenance period (25-45 weeks of age, with normal eggshell color, used to prevent fading and maintain color uniformity): Add 200g of chelated copper-coated probiotic powder and 60mL of emulsified plant essential oil preparation evenly to each ton of complete egg production feed; adopt a step-by-step premixing method, first premix the emulsified plant essential oil preparation with 5kg of corn flour (passed through a 20-mesh sieve), then add the chelated copper-coated probiotic powder and continue mixing for 5min, and finally gradually add the remaining feed, with a total mixing time of not less than 15min; the feeding cycle is 10 days of continuous use and 5 days of discontinuation, with intermittent feeding in a cycle.

[0016] (2) Eggshell color correction period (for laying populations aged ≥55 weeks, late laying period, those with significant color differences in conventional farming, and those with a high incidence of light-shelled / colorful eggs): Add 350g of chelated copper-coated probiotic powder and 120mL of emulsified plant essential oil preparation evenly to each ton of complete laying feed; mix the feed evenly using the above-mentioned step-by-step premixing process, and feed continuously for 15 days without interruption to complete the eggshell color repair and correction. After 15 days, the maintenance program can be switched to the maintenance program depending on the recovery of eggshell color.

[0017] (3) Acute stress relief period (situations such as sudden eggshell discoloration and whitening due to high temperature and heat, flock transfer, vaccination, noise stimulation, etc.): Administer the medication via drinking water. Add 100g of chelated copper-coated probiotic powder and 100mL of emulsified plant essential oil preparation to each ton of clean drinking water, stirring constantly. Withhold water for 1-1.5 hours before administration to induce thirst in the flock, then provide medicated drinking water to ensure concentrated intake of the effective components. Continuous administration for 5-7 days, freshly prepared daily, to quickly reverse stress-induced eggshell discoloration.

[0018] Before use, water stability must be verified: Take 0.3g of copper-encapsulated probiotic powder and 0.1mL of emulsified plant essential oil preparation, add them to 1L of tap water (pH 6.5-7.5, total hardness ≤200mg / L CaCO3), stir gently, and observe after standing for 30 minutes: no oil droplets should be separated from the emulsion, and the probiotic powder should be uniformly suspended without settling. Only after confirming that it is qualified can the product be administered via drinking water.

[0019] If using an automated water supply line, the solution needs to be premixed in the dosing tank (add 30g of powder and 10mL of essential oil per 100L of water, stir for 5 minutes), and then injected into the pipeline through a proportional dosing device.

[0020] S4. Nutritional Enhancement and Aquaculture Management During the aforementioned feeding / watering intervention, feed nutrient parameters are simultaneously adjusted to provide the substrates and coenzymes necessary for protoporphyrin IX synthesis. These nutrients are added during feed production as a premix, or simultaneously mixed into the product of this invention: Ferrous fumarate (iron content 32%), the addition amount is equivalent to 70-90 mg of iron per ton of feed; Vitamin B6 (98% purity), the addition amount is 20-40 mg / ton of feed; Organic zinc (such as zinc methionine or zinc glycine, with a zinc content of 15%) is added at a rate equivalent to 30-50 mg of zinc per ton of feed.

[0021] Meanwhile, maintain the following poultry house management standards: 16 hours / day of light (intensity 10-20 lux), ammonia concentration ≤10 ppm (monitored using an ammonia detector), temperature controlled at 18-25℃, and relative humidity 50-70%. Avoid simultaneous use with antibiotics (especially tetracyclines and fluoroquinolones) and strong disinfectants (such as chlorine-containing disinfectants and peracetic acid) throughout the entire process; the interval between their use should be ≥12 hours to avoid affecting the activity of probiotics and the stability of essential oils.

[0022] In this invention, the product consists of only two ingredients in vitro: a probiotic powder embedded with L-hydroxyproline chelated copper and an emulsified plant essential oil preparation. After entering the digestive tract of poultry, these two ingredients are slowly released and dissociated, forming a targeted regulatory system of probiotics, plant essential oils, and L-hydroxyproline chelated copper in vivo. This system achieves targeted improvement in eggshell color through multi-level biochemical synergy. Specifically: First, the probiotic microcapsules gradually disintegrate in the intestine, releasing embedded L-hydroxyproline chelated copper. The L-hydroxyproline in the chelated copper is decomposed by the probiotic's own extracellular enzymes and converted into a carbon source required for the growth and metabolism of the probiotic, providing energy for the proliferation of the strain and significantly improving the colonization efficiency of the probiotic in the intestinal mucosa. The proliferated compound probiotic can repair the intestinal mucosal barrier, inhibit the reproduction of harmful bacteria in the intestine, reduce the production and entry of inflammatory factors IL-1β and IL-6 into the blood, reduce the damage of systemic inflammation to the shell gland tissue from the source, and create a stable physiological microenvironment for the normal synthesis and deposition of pigments in the shell gland.

[0023] Second, as the chelated copper continues to dissociate, free Cu is released from the system. 2+ The copper ions can undergo in-situ reversible coordination complexation with the phenolic hydroxyl groups contained in components such as oregano oil and rosemary oil in plant essential oils. On the one hand, this enables the slow release of active ingredients in the essential oils in vivo, avoiding rapid metabolic loss of the essential oils and preserving their antioxidant activity for a long time. On the other hand, the complexed products can continuously enhance the antioxidant level of eggshell gland cells and upregulate the activity of ALAS1, the key rate-limiting enzyme in protoporphyrin IX synthesis, thereby promoting the efficient synthesis of protoporphyrin IX from the perspective of enzyme activity.

[0024] Third, the free copper ions obtained from dissociation can also act as key metal coenzymes in the synthesis of protoporphyrin IX, directly participating in the cyclic closure catalytic reaction of porphyrin rings and completing the key catalytic conditions required for pigment synthesis; the L-hydroxyproline released in the system can further participate in the synthesis of collagen in eggshell gland epithelium, repair damaged eggshell gland secretory epithelial tissue, enhance the gland's ability to secrete pigment particles, and improve the uniformity of protoporphyrin IX's adhesion and deposition stability on the eggshell matrix.

[0025] The three components work together in a coordinated manner, acting on three pathways: deposition environment, pigment synthesis, and gland development. Probiotics optimize the internal environment for pigment deposition, essential oils enhance the efficiency of protoporphyrin synthesis, and chelated copper connects the bacterial oil reaction and supplements the key substrates for pigment synthesis. Ultimately, this deepens the eggshell color, reduces individual shell color differences, and provides long-term improvement for problems such as eggshell whitening, excessive color difference, and mottled shells induced by stress in the mid-to-late stages of egg production.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention combines L-hydroxyproline chelated copper with plant essential oils and probiotics for eggshell color regulation, constructing a ternary synergistic system that includes intestinal flora regulation, uterine inflammation inhibition, and pigment synthase activation. Experiments show that, compared with using probiotics and essential oils (without added chelated copper) or using chelated copper alone, the combination of the three increases the eggshell a value (redness) by more than 12% and reduces the proportion of light-shelled eggs by more than 50%.

[0027] (2) In this invention, L-hydroxyproline chelated copper is encapsulated inside probiotic microcapsules. The extracellular enzymes of probiotics decompose L-hydroxyproline into metabolic carbon sources, promoting probiotic colonization in the intestine, while simultaneously achieving the sustained release of copper ions. This design solves the problem of copper ions reacting and becoming inactive with the phenolic hydroxyl groups of essential oils during storage (activity retention ≥90% after 12 months of storage) and significantly improves the survival rate of probiotics through the stomach (≥85%).

[0028] (3) The present invention provides a differentiated application scheme for different physiological stages. It sets the optimal addition amount and feeding / drinking route for three typical scenarios: maintaining peak egg production, correcting old laying hens, and emergency treatment of acute stress. Compared with the existing fixed dose continuous addition method, it can reduce the total dosage by 20-30% and shorten the stress fading recovery time to 5-7 days (normally it takes 10-14 days).

[0029] (4) This invention uses water-in-oil emulsified essential oil and drinking water administration technology, which solves the problem that essential oil is insoluble in water and difficult to administer evenly through drinking water. The emulsified essential oil droplets have a particle size of ≤200nm and can be stably dispersed in drinking water for more than 24 hours. Combined with the water withdrawal induction method, it ensures that the flock can quickly ingest an effective dose, which is especially suitable for emergency intervention in stressful situations.

[0030] (5) This invention clarifies the nutritional synergy scheme and aquaculture control parameters, provides substrates for protoporphyrin IX synthesis such as iron, vitamin B6, and zinc, and limits conditions such as light, ammonia, and antibiotic intervals, so that the technical effect of this invention is highly reproducible and can be implemented by ordinary farms without additional exploration of conditions.

[0031] The present invention will be explained in detail below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0032] Figure 1 A flowchart illustrating the steps of the method for controlling the color of poultry eggshells provided by the present invention; Figure 2 This is a line graph showing the dynamic changes of the value of a in Embodiment 1, Comparative Example 1, and Comparative Example 2 of the present invention. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more comprehensive description of the present invention will be given below with reference to the accompanying drawings, which illustrate several embodiments of the present invention. However, the present invention can be implemented in different forms and is not limited to the embodiments described in the text. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Example 1: This example is for Hy-Line Brown laying hens aged 25-45 weeks during their peak egg-laying period, and is used to prevent eggshell fading and maintain uniform coloring.

[0036] Please refer to the attached document carefully. Figure 1 A method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics includes the following steps: 1. Preparation of microcapsule-coated compound probiotic powder containing L-hydroxyproline-chelated copper. (1) Preparation of compound probiotic bacterial solution: 35% *Lactobacillus plantarum*, 25% *Bacillus subtilis*, 20% *Enterococcus faecalis*, and 20% *Saccharomyces cerevisiae* were prepared by mass percentage. After activation, *Lactobacillus plantarum* and *Enterococcus faecalis* were inoculated into MRS liquid medium at 5% (v / v) of the total inoculation amount, *Bacillus subtilis* into LB liquid medium, and *Saccharomyces cerevisiae* into YPD liquid medium. The cultures were incubated at 37℃ for 24 h. The bacterial cells were collected by centrifugation, mixed, washed twice with sterile physiological saline, and resuspended to a bacterial concentration of 1×10⁻⁶. 10 The CFU / mL (plate count method) was used to obtain the compound bacterial solution.

[0037] (2) Adding L-hydroxyproline chelated copper: Weigh L-hydroxyproline chelated copper powder (12% effective chelated copper content, 88% L-hydroxyproline carrier content) at 17% of the dry weight of the bacterial cells in the compound bacterial solution. Pre-calibration of bacterial dry weight: Take 10 mL of the compound bacterial solution and dry it at 105℃ to constant weight, and measure the dry weight as 5.2 mg / mL. Slowly add the chelated copper powder and stir to dissolve evenly.

[0038] (3) Preparation of microcapsule coating wall material: Sodium alginate (viscosity 300 mPa·s) and maltodextrin (DE value 18) were mixed at a mass ratio of 1:2, and deionized water was added to prepare a wall material solution with a total concentration of 6% (w / v). The solution was sterilized at 121℃ for 15 min and then cooled to room temperature.

[0039] (4) Preparation of microcapsules with embedded chelated copper: The above-mentioned composite bacterial solution with added chelated copper was mixed with the wall material solution at a core material: wall material ratio of 1:2 (volume ratio), and stirred to disperse evenly. A BUCHIB-290 spray dryer was used, with the inlet air temperature set at 162℃, the outlet air temperature at 81℃, the atomization pressure at 0.2MPa, the feed rate at 40mL / min, and the nozzle diameter at 0.7mm. The dried powder was collected and passed through an 80-mesh sieve to obtain probiotic powder with embedded chelated copper. The total number of viable bacteria was tested to be 5.6×10⁻⁶. 9 CFU / g, gastric survival rate 88% [Assay method: Take 1g of sample and add 50mL of artificial gastric fluid (pH2.0, containing 3.2g / L pepsin), shake at 37℃ for 2h, take samples, spread them and count the survival rate], moisture content 7.2%, viable bacteria attenuation rate 4.2% / month after 12 months of sealed storage at room temperature.

[0040] 2. Preparation of emulsified plant essential oil formulations (1) Compound essential oil stock solution: Take 50% oregano oil, 30% rosemary oil, 12% cinnamon oil and 8% thyme oil by volume percentage and mix them evenly at room temperature.

[0041] (2) Preparation of the emulsion system: Based on 100 parts of the final emulsion, take 80 parts of deionized water, add 8 parts of food-grade soybean lecithin and 2 parts of Tween-80, and stir in a 40℃ water bath until completely dissolved to form an aqueous phase. Under high-speed shearing conditions (9000r / min), slowly add 10 parts of the compound essential oil mother liquor (water phase to oil phase mass ratio 9:1), and continue shearing for 10 min after the addition is complete. Let stand at room temperature for 30 min to defoam, and obtain a water-in-oil emulsion plant essential oil preparation. After testing, after 6 months of sealed storage at 25℃ away from light, the effective active ingredient retention rate was 96.5% (gas chromatography determination of carvacrol and thymol content), with no stratification or precipitation.

[0042] 3. Apply in stages For 25-week-old Hy-Line Brown laying hens (94% egg production rate, normal eggshell color), add 200g of the above-mentioned copper-embedded probiotic powder and 60mL of emulsified plant essential oil preparation evenly to each ton of complete laying feed. Use a step-by-step premixing method: first, premix the emulsified essential oil with 5kg of corn flour (passed through a 20-mesh sieve) for 5 minutes, then add the powder and continue mixing for 5 minutes, finally gradually adding it to the remaining feed, with a total mixing time of 18 minutes. Feeding cycle: 10 days of continuous use, followed by a 5-day break, with intermittent feeding. Simultaneously, add ferrous fumarate (equivalent to 80mg / ton of iron), vitamin B6 (30mg / ton), and zinc methionine (equivalent to 40mg / ton of zinc) to the feed. Poultry house management: 16h / day light (intensity 15lux), ammonia concentration ≤8ppm, temperature 22℃, humidity 60%, and avoid antibiotics throughout the entire process.

[0043] Comparative Example 1 (Blank Control): This example differs from Example 1 in that it does not contain embedded copper-coated probiotic powder, emulsified plant essential oil preparations, or additional ferrous fumarate, vitamin B6, or organic zinc. Only a basic complete egg-laying diet is fed, and no additives are added to the drinking water. The species, rearing period, and testing indicators are the same as in Example 1.

[0044] Comparative Example 2 (lacking chelated copper): The difference from Example 1 is the use of microcapsule-coated compound probiotic powder without added L-hydroxyproline chelated copper (probiotics and microcapsule coating are the same as in Example 1), and the same emulsified plant essential oil preparation as in Example 1, with the simultaneous addition of ferrous fumarate (iron 80 mg / ton), vitamin B6 (30 mg / ton), and zinc methionine (zinc 40 mg / ton). The dosage, feeding cycle, and target animals were the same as in Example 1 (10 days of continuous use, followed by a 5-day break, repeated twice).

[0045] One hundred 25-week-old Hy-Line Brown laying hens were selected for each group, with identical rearing environment and basal diet. Egg samples were collected on day 0 (before the start of the experiment), day 10 (end of the first cycle), day 20 (middle of the second cycle), and day 30 (end of the second cycle), with 30 eggs randomly selected from each group for testing. Testing indicators: (1) Eggshell a value (redness): The CR-400 colorimeter with a standard D65 light source was used to measure the equatorial part of the eggshell. Three points were measured for each egg and the average value was taken. (2) Proportion of light-shelled eggs: The Roche eggshell color chart (1-15 grades) is used. Eggs with a grade of ≤5 are judged to be light-shelled. (3) Eggshell color uniformity (color difference ΔE): Three points are measured at equal intervals at the equator of each egg. The color difference ΔE of CIELaB is calculated. Then the standard deviation of ΔE of the 30 eggs in the group is calculated. The smaller the standard deviation, the more uniform the color. (4) Egg production rate: Record the number of eggs produced daily and calculate the weekly average egg production rate; (5) Survival rate of probiotics after passing through the stomach: Take 1g of powder, treat with artificial gastric juice for 2 hours, and then spread and count.

[0046] The test results are shown in Tables 1 and 2. Figure 2 As shown.

[0047] Table 1. Statistics of key indicators for day 0 and day 30.

[0048] Table 2 Dynamic changes of a value at each stage

[0049] Comparing Example 1 with Comparative Example 1, the eggshell a value of Example 1 reached 14.8 after 30 days, an increase of 22.3% compared to Comparative Example 1. The a value of Comparative Example 1 remained essentially unchanged over 30 days (12.1–12.3), indicating that eggshell color naturally remained stable during peak egg production without intervention. The proportion of light-shelled eggs in Example 1 decreased from 4.2% to 1.1%, a reduction of 73.8%; while in Comparative Example 1, it remained around 4.5%, with no significant change. The standard deviation of color difference ΔE in Example 1 decreased from 2.1 to 1.0, an improvement of 52.4%; while in Comparative Example 1, there was no significant improvement (2.1→2.2). The egg production rate of Example 1 slightly increased (94.2%→95.6%), while in Comparative Example 1 it slightly decreased (94.2%→93.8%), indicating that the product of this invention has no negative impact on production performance, and may even have a slight promoting effect. Therefore, this invention is significantly superior to the blank control group without any added product in maintaining eggshell color and uniformity during peak egg production.

[0050] Comparing Example 1 and Comparative Example 2, the a-value of Example 1 on day 10 was 13.9, an increase of 0.9 units (relative improvement of 6.9%) compared to Comparative Example 2 (13.0); on day 30, Example 1 showed an increase of 1.4 units (relative improvement of 10.4%) compared to Comparative Example 2. The differences were statistically significant. On day 30, the proportion of light-shelled eggs in Example 1 was 1.1%, a decrease of 52.2% compared to Comparative Example 2 (2.3%). The standard deviation of color difference ΔE in Example 1 was 1.0, an improvement of 33.3% compared to Comparative Example 2 (1.5). The survival rate of probiotics after gastric passage in both Example 1 and Comparative Example 2 was between 87-88%, with no significant difference, indicating that the addition of chelated copper did not adversely affect the survival rate of microencapsulated probiotics. Compared to Comparative Example 2, Example 1, which introduced L-hydroxyproline chelated copper into the probiotic-essential oil binary system, further improved eggshell redness and significantly reduced the proportion of light-shelled eggs. This indicates that: Chelated copper, by participating in protoporphyrin IX synthesis (as a copper coenzyme) and promoting the repair of eggshell gland epithelial tissue with L-hydroxyproline, produces additional synergistic effects on the basis of the binary system. The same level of improvement cannot be achieved by relying solely on probiotics combined with essential oils and common trace elements (iron, zinc, B6); targeted copper supplementation is irreplaceable.

[0051] Furthermore, regarding the tolerability of the intermittent feeding pattern, as shown in Table 2, the a value on day 20 (5 days after 5 days of discontinuation) was 13.5, slightly lower than the 13.9 at the end of day 10, but still significantly higher than the baseline (12.3), and there was no sharp drop in color during the discontinuation period. The a value rose again to 14.8 on day 30 after resumption of use, indicating that the regimen has good effect maintenance and restart responsiveness. In contrast, the a value in Comparative Example 2 decreased from 13.0 to 12.6 during the discontinuation period, a larger decrease than in Example 1, further demonstrating that the presence of chelated copper helps prolong the color maintenance time during the discontinuation period.

[0052] In summary, the method described in Embodiment 1 of this invention is significantly superior to the blank control and the binary system lacking chelated copper in maintaining eggshell color during peak egg production. The addition of chelated copper not only directly enhances pigment synthesis but also prolongs the color maintenance time during intermittent drug withdrawal, demonstrating the superiority of the ternary synergistic effect of this invention.

[0053] Example 2: This example is for 58-week-old Lohmann Pink laying hens in the late laying period, where there has been obvious eggshell discoloration and an increased proportion of light-shelled eggs (about 15%).

[0054] A method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics includes the following steps: 1. Preparation of microcapsule-coated compound probiotic powder containing L-hydroxyproline-chelated copper. (1) Preparation of compound probiotic bacterial solution: 38% *Lactobacillus plantarum*, 22% *Bacillus subtilis*, 18% *Enterococcus faecalis*, and 22% *Saccharomyces cerevisiae* were prepared by mass percentage. The bacterial strains were activated, cultured, centrifuged, and washed as in Example 1, and resuspended to a bacterial concentration of 1.2 × 10⁻⁶. 10 CFU / mL.

[0055] (2) Add L-hydroxyproline chelated copper: Weigh out chelated copper powder as described in Example 1 at 18% of the dry weight of the bacterial cells. Dry weight determination of bacterial cells: 10 mL of bacterial solution was dried to obtain a dry weight of 5.5 mg / mL. Add slowly and stir to dissolve.

[0056] (3) Microcapsule coating: The wall material preparation is the same as in Example 1. Spray drying parameters are: inlet air temperature 165℃, outlet air temperature 83℃, atomization pressure 0.22MPa, and feed rate 35mL / min. The resulting powder has a total viable count of 5.2×10⁻⁶. 9 CFU / g, gastric survival rate 86%, moisture content 7.8%, monthly decay rate 4.5%.

[0057] 2. Preparation of emulsified plant essential oil formulations (1) Compound essential oil mother liquor: Take 48% oregano oil, 32% rosemary oil, 10% cinnamon oil and 10% thyme oil by volume percentage.

[0058] (2) Emulsion system: 78 parts deionized water, 9 parts soybean lecithin, and 3 parts Tween-80 per 100 parts were dissolved at 40℃. 10 parts essential oil mother liquor were slowly added under high-speed shearing at 9500 r / min and sheared for 9 min. The resulting emulsion had a 6-month retention rate of 95.2%.

[0059] 3. Apply in stages Add 350g of copper-encapsulated probiotic powder and 120mL of emulsified essential oil to each ton of feed, premixing stepwise (method as in Example 1), and feed continuously for 15 days. Simultaneously, add ferrous fumarate (iron 90mg / ton), vitamin B6 (35mg / ton), and zinc glycine (zinc 45mg / ton) to the feed. Poultry house management is the same as in Example 1.

[0060] Comparative Example 3 (Blank Control): This example differs from Example 2 in that it does not contain the added chelated copper-coated probiotic powder, emulsified plant essential oil preparation, or additional ferrous fumarate, vitamin B6, or organic zinc. Only a basic complete egg-laying diet is fed, and no additives are added to the drinking water. The species, rearing period, and testing indicators are the same as in Example 2.

[0061] Comparative Example 4 (Positive Control of Copper Methionine): 100 Lohmann Pink laying hens of the same batch and age (58 weeks) as in Example 2 were selected. The rearing environment, basal diet, lighting, temperature and humidity, and immunization program were completely identical to the experimental group. This control group used feed-grade copper methionine (DL-copper methionine, copper content 16%) as the copper source, aiming to achieve the same copper content in the feed as the experimental group, i.e., 175 mg / ton. Calculations showed that approximately 1.09 grams of copper methionine were needed per ton of feed. Due to the extremely small amount, a three-stage premixing method was required: first, 1.09 grams of copper methionine was premixed with 100 grams of corn flour (passed through a 60-mesh sieve) in a small mixer for 10 minutes; then, this premix was mixed with 1 kg of corn flour for 10 minutes; finally, it was gradually mixed with the remaining feed, with a total mixing time of no less than 15 minutes to ensure uniform distribution.

[0062] In addition to the copper source, the control group was simultaneously supplemented with the same doses of ferrous fumarate (90 mg / ton), vitamin B6 (35 mg / ton), and zinc glycine (45 mg / ton) as the experimental group, but without the copper-coated probiotic powder and emulsified plant essential oil preparation of this invention. The feeding method was the same as in Example 2, with continuous feeding for 15 days.

[0063] After 15 days of feeding, Example 2, Comparative Example 3, and Comparative Example 4 showed significant differences in eggshell color indicators: In Comparative Example 3, the proportion of light-shelled eggs increased slightly from 15.3% before the experiment to 16.2%, and the eggshell a value (redness) changed from 9.8 to 10.1, remaining basically the same; the standard deviation of color difference ΔE remained at around 3.1, indicating that under no intervention conditions, the eggshell color of older laying hens further deteriorated or at least could not recover naturally.

[0064] In Comparative Example 4, the proportion of light-shelled eggs decreased to 8.5%, the a value increased to 11.9, and the standard deviation of color difference ΔE decreased to 2.2, indicating that supplementing with copper methionine alone has a certain positive effect on eggshell color, which is consistent with its function as a coenzyme for protoporphyrin IX synthesis.

[0065] In Example 2 of this invention, the proportion of light-shelled eggs was further reduced to 4.1%, which is 74.7% lower than that of Comparative Example 3 and 51.8% lower than that of Comparative Example 4; the a value reached 13.5, which is 33.7% higher than that of Comparative Example 3 and 13.4% higher than that of Comparative Example 4; the standard deviation of color difference ΔE was reduced to 1.4, and the improvement in color uniformity was significantly better than that of Comparative Example 3 and Comparative Example 4.

[0066] Therefore, it can be concluded that, under the premise of equal copper addition, the effect of the present invention is far superior to the control group that simply adds copper methionine. Since the difference between Example 2 and Comparative Example 4 lies primarily in the experimental group containing additional coated compound probiotics and compound emulsified plant essential oils, this can be clearly attributed to the synergistic effect of probiotics and plant essential oils on copper ions. Specifically, probiotics improve intestinal health and the uterine microecological environment, while plant essential oils reduce oxidative stress and inflammatory responses in the shell glands. Together, they create favorable conditions for the efficient participation of copper ions in protoporphyrin IX synthesis, resulting in a stronger color-enhancing effect with the same dose of copper. Furthermore, L-hydroxyproline chelated copper itself may have higher bioavailability than copper methionine, which may also be one of the reasons for the superior effect of the experimental group. In summary, the scheme of Example 2 of the present invention is significantly superior to the blank control group without any added products and the control group with equal copper amounts of copper methionine in correcting eggshell color in aged eggs, fully demonstrating the synergistic regulatory advantages of the ternary system of probiotics combined with plant essential oils and L-hydroxyproline chelated copper.

[0067] Example 3: This example addresses the sudden whitening and discoloration of eggshells in Hy-Line Grey hens, resulting in an 8% decrease in egg production, caused by high summer temperatures (above 35°C).

[0068] A method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics includes the following steps: 1. Preparation of microcapsule-coated compound probiotic powder containing L-hydroxyproline-chelated copper. (1) Preparation of compound probiotic bacterial solution: 32% *Lactobacillus plantarum*, 28% *Bacillus subtilis*, 22% *Enterococcus faecalis*, and 18% *Saccharomyces cerevisiae* were prepared by weight percentage. The strain activation, cultivation, centrifugation, and washing were the same as in Example 1. The bacterial solution concentration was 1.0 × 10⁻⁶. 10 CFU / mL.

[0069] (2) Add L-hydroxyproline chelated copper: Weigh out chelated copper powder at 16% of the dry weight of the bacterial cells. Dry weight determination of bacterial cells: 10 mL of bacterial solution was dried to obtain a dry weight of 5.0 mg / mL. Add slowly and stir to dissolve.

[0070] (3) Microcapsule coating: The wall material preparation is the same as in Example 1. Spray drying parameters are: inlet air temperature 160℃, outlet air temperature 80℃, atomization pressure 0.18MPa, and feed rate 45mL / min. The resulting powder has a total viable count of 5.8 × 10⁻⁶. 9 CFU / g, gastric survival rate 89%.

[0071] 2. Preparation of emulsified plant essential oil formulations (1) Compound essential oil mother liquor: oregano oil 52%, rosemary oil 28%, cinnamon oil 14%, thyme oil 6%.

[0072] (2) Emulsion system: 82 parts deionized water, 7 parts soybean lecithin, 1 part Tween-80, shear speed 8500 r / min, shear for 10 min. Emulsion is obtained.

[0073] 3. Apply in stages (through drinking water) First, verify the stability in drinking water: Add 0.3g of powder and 0.1mL of essential oil to 1L of tap water (pH 7.2, total hardness 150mg / L CaCO3), stir and let stand for 30min. No oil droplets were separated from the emulsion, the powder was uniformly suspended, and no flocculation was observed.

[0074] Add 100g of powder and 100mL of essential oil to each ton of drinking water, stirring constantly. Withhold water for 1.5 hours before administering the medication, then provide the medicated water, which should be consumed within 2 hours. Continue this treatment for 6 days, preparing fresh solutions daily. Simultaneously, add ferrous fumarate (70mg / ton iron), vitamin B6 (25mg / ton), and organic zinc (35mg / ton zinc) to the feed. Poultry house management is the same as in Example 1.

[0075] Comparative Example 5 (Blank Control): This example differs from Example 3 in that it does not contain embedded copper-coated probiotic powder, emulsified plant essential oil preparations, or additional ferrous fumarate, vitamin B6, or organic zinc. Only a basic complete egg-laying diet is fed, and no additives are added to the drinking water. The species, rearing period, and testing indicators are the same as in Example 3.

[0076] Comparative Example 6 (Fixed-Dose Water Control): Under the same high-temperature stress environment, the same water administration route as in Example 3 was used, but the dosage was the maintenance dose (i.e., 50 grams of copper-encapsulated probiotic powder and 60 ml of emulsified plant essential oil preparation per ton of drinking water). Water was withheld for 1.5 hours before administration, and the treatment was continued for 6 consecutive days, with fresh preparation daily. Iron, B6, and zinc were simultaneously supplemented in the feed (same as in Example 3). Thus, the only difference between Comparative Example 6 and Example 3 was the dosage; the administration route and operation method were completely identical.

[0077] After 6 days of treatment, the differences in results among the groups were significant: In Comparative Example 5, the whitening of eggshells continued to worsen, with the proportion of light-shelled eggs increasing from 18.6% after stress to 21.3%, the eggshell a value (redness) further decreasing from 9.2 to 8.7, and the egg production rate decreasing from 82% to 79%. This indicates that if no intervention measures are taken under continuous high-temperature stress, eggshell color and production performance will continue to deteriorate.

[0078] In Comparative Example 6, the proportion of shallow-shelled eggs dropped to 12.5%, the a value rebounded to 10.4, and the egg production rate recovered to 85%, but the effect was slow, and no obvious change was observed until the 4th day.

[0079] This invention significantly improved eggshell whitening on the 3rd day after administration, and by the 6th day, the proportion of light-shelled eggs decreased to 3.2%, the a value recovered to 13.8 (close to pre-stress levels), and the egg production rate rebounded to 91%. Compared with Comparative Example 6, Example 3 showed a 3-day shorter time to effectiveness, a 74.4% reduction in the proportion of light-shelled eggs, and a 32.7% increase in the a value. Since the administration routes and operation methods were exactly the same in both groups, the difference in effect could only be attributed to the increased dosage during the emergency period. This indicates that in emergency situations where acute stress causes rapid eggshell discoloration, appropriately increasing the dosage and using a rapid drinking water route can significantly accelerate the recovery of eggshell color and achieve better improvement results. Meanwhile, the continued deterioration in Comparative Example 5 also confirms that the self-recovery ability of stress-induced discoloration is limited and active intervention is necessary. Therefore, the scheme in Example 3 provides a practical and effective emergency measure for farms to cope with sudden stress events such as high temperature, relocation, and immunization.

[0080] In summary, this invention embeds L-hydroxyproline-chelated copper into a compound probiotic microcapsule, which is then used in conjunction with a compound emulsified plant essential oil. Differentiated dosages and administration routes are employed for three physiological stages: maintaining peak egg production, correcting eggshell color, and providing emergency treatment during acute stress. Through the synergistic effect of probiotics, essential oils, and copper ions, rapid improvement and long-term maintenance of eggshell color are achieved. Examples demonstrate that this invention is significantly superior to existing technologies in improving eggshell redness, reducing the proportion of light-shelled eggs, and shortening stress recovery time.

[0081] The present invention has been described by way of example in conjunction with the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvement made by adopting the inventive concept and technical solution of the present invention, or the direct application of the inventive concept and technical solution of the present invention to other occasions without modification, shall be within the protection scope of the present invention.

Claims

1. A method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics, characterized in that: Apply powder A and formulation B in the following phased manner: (1) During the peak egg production period: Add 200g of powder A and 60mL of preparation B to each ton of feed; (2) Eggshell color correction period: Add 350g of the A powder and 120mL of the B preparation to each ton of feed; (3) Acute stress emergency treatment period: Add 100g of powder A and 100mL of preparation B to each ton of drinking water; The A powder is a microcapsule-coated compound probiotic powder with L-hydroxyproline chelated copper, and the B preparation is a water-in-oil type compound emulsified plant essential oil preparation. The microcapsule-coated compound probiotic powder containing L-hydroxyproline chelated copper contains Lactobacillus plantarum, Bacillus subtilis, Enterococcus faecalis and Saccharomyces cerevisiae. The water-in-oil type compound emulsified plant essential oil preparation contains oregano oil, rosemary oil, cinnamon oil and thyme oil.

2. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: The compound probiotic contains 30-40% Lactobacillus plantarum, 20-30% Bacillus subtilis, 15-25% Enterococcus faecalis, and 15-25% Saccharomyces cerevisiae.

3. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: In the compound essential oil, oregano oil accounts for 45-55%, rosemary oil accounts for 25-35%, cinnamon oil accounts for 8-16%, and thyme oil accounts for 5-12%.

4. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: When preparing the A powder, L-hydroxyproline chelated copper powder is added at 15%-20% of the dry weight of the compound probiotic cells; then the compound bacterial solution with added L-hydroxyproline chelated copper is mixed evenly with the microcapsule coating wall material solution at a volume ratio of 1:2, and microencapsulated by low-temperature spray drying to obtain the microcapsule-coated compound probiotic powder with embedded L-hydroxyproline chelated copper.

5. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 4, characterized in that: The microcapsule coating wall material solution is prepared by mixing sodium alginate and maltodextrin at a mass ratio of 1:2 and then adding deionized water.

6. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: The total viable bacteria count of powder A is ≥5×10⁻⁶. 9 CFU / g, gastric survival rate ≥85%, moisture content ≤8%.

7. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: When preparing formulation B, the raw materials of the compound essential oil are mixed evenly to obtain the compound essential oil mother liquor; then, under high-speed shearing conditions, the compound essential oil mother liquor is added to the aqueous phase, with the mass ratio of the aqueous phase to the oil phase being 9:

1. After mixing evenly, the mixture is allowed to stand at room temperature to defoam, thus obtaining the water-in-oil type compound emulsified plant essential oil preparation.

8. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 7, characterized in that: The aqueous phase comprises 75-85 parts of deionized water, 6-10 parts of food-grade soybean lecithin, and 1-3 parts of Tween-80.

9. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: The feeding cycle for the peak egg production maintenance period is 10 consecutive days followed by 5 days of discontinuation, with intermittent feeding in a cyclical manner. The eggshell color correction period involves continuous feeding for 15 days. During the acute stress emergency period, drink water continuously for 5-7 days.

10. The method for synergistic regulation of poultry eggshell color by plant essential oils and probiotics according to claim 1, characterized in that: During feeding or water intervention, ferrous fumarate 70-90 mg / ton, vitamin B6 20-40 mg / ton and organic zinc 30-50 mg / ton should be added to the feed simultaneously.

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