Use of astaxanthin fermented by phaffia rhodozyma in preparation of composition for improving follicular development of egg chicken in post-egg production period and improving egg quality

Abstract

The application relates to the field of livestock breeding technology, and discloses application of red phaffia rhodozyma fermented astaxanthin in preparation of a composition for improving follicle development of egg hens in a post-egg production period and improving egg quality, and a feed composition containing the astaxanthin. The red phaffia rhodozyma fermented astaxanthin is added into feed of the egg hens in the post-egg production period in an adding amount of 500-2000 g / t, and continuous feeding is not less than three weeks. The application solves the problems of poor follicle development of the egg hens in the post-egg production period caused by ovary function recession and decreased antioxidant quality of the eggs, can improve grade follicle development, increase large white follicle reserves, significantly improve total antioxidant capacity of egg yolk and astaxanthin deposition, selectively improve egg yolk flavor, improve high-temperature storage stability of the eggs, and greatly reduce oxidative stress damage to the ovaries of the egg hens.

Description

Technical Field

[0001] This application relates to the field of animal husbandry technology, and in particular to the application of red phaf yeast fermented astaxanthin in the preparation of a composition for improving follicle development and egg quality in laying hens during the late laying period, a feed composition containing the astaxanthin, and functional eggs. Background Technology

[0002] As laying hens enter the later stages of egg production (usually after 60 weeks of age), ovarian function naturally declines, leading to insufficient replenishment of large white follicles and slow development and reduced diameter of graded follicles, resulting in a decrease in sustained egg production. Simultaneously, the quality of eggs produced by hens in the later stages of egg production also declines, manifesting as insufficient antioxidant capacity in the yolk, deteriorated flavor, and rapid quality deterioration during high-temperature distribution in summer.

[0003] In existing technologies, methods such as light regulation, hormone intervention, vitamin E supplementation, or synthetic colorants are commonly used to prolong the laying cycle of hens and improve egg quality. However, these methods may have limited effectiveness or pose food safety risks. For example, while the synthetic colorant canthaxanthin can improve egg yolk color, related studies have shown that it can lead to a significant decrease in T-SOD activity in the ovaries of laying hens, causing oxidative stress damage to the reproductive system.

[0004] Therefore, it is necessary to develop a technical solution that can specifically improve follicle development in laying hens during the later stages of egg production, and simultaneously enhance egg quality and flavor. Summary of the Invention

[0005] This application provides the application of red phaf yeast fermented astaxanthin in the preparation of a composition for improving follicle development in laying hens during the late laying period and / or enhancing the endogenous antioxidant quality of eggs, a feed composition containing the astaxanthin, and functional eggs, which solves the technical problems of poor follicle development and decreased egg quality caused by the decline in ovarian function in laying hens during the late laying period.

[0006] In one aspect, this application provides the use of red phaf yeast fermented astaxanthin in the preparation of a composition for improving follicle development in late-laying hens and enhancing egg quality.

[0007] Furthermore, the effective content of astaxanthin in the astaxanthin fermented by the red phaf yeast is 1-2%.

[0008] Furthermore, the amount of astaxanthin fermented by the red phaf yeast added to the feed is 500-2000 g / t.

[0009] Furthermore, the amount of astaxanthin fermented by Pharfogel's yeast added to the feed is 500-1000 g / t; or the amount of astaxanthin fermented by Pharfogel's yeast added to the feed is 1000-2000 g / t.

[0010] Furthermore, the continuous feeding period for the astaxanthin fermented by the red phaf yeast is no less than three weeks.

[0011] Secondly, this application provides a composition for improving follicle development in laying hens during the later stages of egg production and enhancing egg quality, comprising astaxanthin fermented with red phaf yeast and excipients.

[0012] Thirdly, this application provides a functional egg produced by feeding the above-mentioned composition, wherein the functional egg has a T-AOC ≥ 0.50 μM / prot and an astaxanthin deposition in the yolk ≥ 70 μg / g.

[0013] Through one or more embodiments of the above embodiments of the present invention, at least the following technical effects can be achieved: This application involves adding astaxanthin fermented with Pharbaugh yeast to the feed of laying hens in the late laying period. By utilizing the strong free radical scavenging ability of the 100% dextrorotatory configuration (3R-3'R) of astaxanthin derived from Pharbaugh yeast, as well as the synergistic nutritional effects of polysaccharides, nucleotides, and other components in the yeast matrix, astaxanthin is efficiently digested and absorbed by laying hens and deposited in the egg yolk. This allows it to simultaneously play a role in three dimensions: improving follicle development in laying hens in the late laying period, enhancing the endogenous antioxidant quality of eggs, and optimizing the flavor of egg yolks.

[0014] Specifically, in terms of follicle development, astaxanthin fermented with red phloxetine can protect the normal development of follicle cells by clearing free radicals in ovarian tissue, reducing lipid peroxidation damage, increasing the number of large white follicles, and promoting the increase of graded follicle diameter. Unlike cantharidin, which causes a significant decrease in ovarian T-SOD, the ovarian T-SOD of each astaxanthin-added group remained within the normal range, demonstrating a fundamental advantage in safety.

[0015] In terms of egg quality, astaxanthin deposited in egg yolks significantly improves the total antioxidant capacity (T-AOC) of egg yolks, and this effect can reach the saturation threshold at a low addition amount, allowing farmers to obtain maximum antioxidant quality improvement at a low cost. At the same time, the antioxidant effect of astaxanthin effectively inhibits the abnormal oxidation of egg yolk lipids, reduces the bitterness and astringency of egg yolks, enhances umami flavor, and achieves selective improvement of egg yolk flavor.

[0016] In addition, the astaxanthin deposited in the egg yolk forms an endogenous antioxidant protection system, which can effectively delay the deterioration of egg quality such as protein hydration and yolk scattering during the high-temperature circulation process in summer, and improve the high-temperature storage stability of eggs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a bar chart showing the effect of adding different doses of astaxanthin to the diet on the ovarian weight of laying hens in the embodiments of this application.

[0019] Figure 2 This is a bar chart showing the distribution of egg freshness grades after 7 days of storage in an embodiment of this application.

[0020] Figure 3 This is a radar diagram showing the electronic nose response of each group of egg yolks (A) and albumen (B) in the embodiments of this application. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Furthermore, the character " / " in this document, unless otherwise specified, generally indicates that the preceding and following related objects have an "or" relationship.

[0023] In the description of this invention, the term "late laying period" refers to the physiological stage in which the egg production rate of laying hens begins to decline continuously, typically corresponding to ages over 60 weeks. The term "graded follicles" refers to mature follicles with a diameter greater than 9 mm that are arranged in a grade (usually labeled F1-F5, with F1 being the largest). The term "large white follicles" refers to follicle reserves with a diameter of 2-5 mm. The term "supplementary feed" refers to feed components other than astaxanthin fermented with Pharrellis rubrum, including but not limited to energy feeds, protein feeds, minerals, vitamin premixes, and functional additives.

[0024] As described in the background section, laying hens in the later stages of egg production face systemic problems such as declining ovarian function, insufficient antioxidant quality of eggs, and deterioration in flavor. Existing methods such as light regulation, hormone intervention, vitamin E supplementation, and canthaxanthin coloring agents have limited effectiveness or pose safety risks.

[0025] To address the above problems, this application provides a new technical approach: Natural astaxanthin produced by the fermentation of Pharbitis rubescens was selected as the source of functional composition and combined with excipients to prepare feed composition; Feeding this feed composition to late-laying hens allows astaxanthin to be deposited in the yolk through the hens' digestive and absorption pathways, thereby exerting multi-dimensional biological functions to improve follicle development and enhance the endogenous antioxidant quality of eggs. After feeding continuously for no less than three weeks, functional eggs with T-AOC ≥ 0.50 μM / prot and astaxanthin deposition in the yolk ≥ 70 μg / g can be produced.

[0026] Astaxanthin (ASTA) is a ketocarotenoid formed by the oxidation of β-carotene, with the molecular formula C40H52O4. Its molecular structure features a terminal ring containing both a hydroxyl and a ketone group on each side of the conjugated polyene chain. This unique structure gives astaxanthin both hydrophilic and lipophilic ends, enabling it to penetrate the cell membrane bilayer and scavenge free radicals both inside and outside the membrane.

[0027] Red Pharbour yeast is a single-celled yeast. After its cell wall is broken, the yeast polysaccharides, nucleic acids, nucleotides and amino acids contained in the yeast itself can enhance nutrient digestion and absorption and metabolic synergy, which is beneficial to the absorption and conversion of astaxanthin in poultry.

[0028] In traditional methods, the astaxanthin conversion rate using Haematococcus pluvialis or its extracts is typically between 2% and 5%. However, the conversion rate of astaxanthin derived from Pharfogel's yeast can be further improved, and the raw material cost is lower. Of course, within the technical framework of this application, astaxanthin products fermented from other batches or sources of Pharfogel's yeast can also be used, as long as their effective astaxanthin content is within the range of 1% to 2%.

[0029] In some embodiments, the effective content of astaxanthin can be any value or a range between any two of 1.0%, 1.2%, 1.5%, 1.8%, and 2.0%.

[0030] In some embodiments, the amount of astaxanthin fermented with Pharbitis rubescens added to feed can be any value or a range between any two of the following: 500 g / t, 750 g / t, 1000 g / t, 1500 g / t, and 2000 g / t.

[0031] If the addition amount is less than 500 g / t, the deposition of astaxanthin in the egg yolk may not be sufficient to reach a saturation plateau, resulting in limited improvement in antioxidant capacity. If the addition amount is higher than 2000 g / t, it will not only fail to provide significant functional gains but also increase feed costs. Therefore, this application recommends an addition range of 500-2000 g / t.

[0032] In some embodiments, the supplementary feed can be a corn-soybean meal type basal diet, a corn-mixed meal type basal diet, or a wheat-soybean meal type basal diet, as long as it meets the basic nutritional needs of laying hens in the late laying period. The metabolizable energy in the supplementary feed can be any value or a range between any two of 2500, 2550, 2601, 2650, and 2700 kcal / kg; the crude protein content can be any value or a range between any two of 15.0%, 15.5%, 15.78%, 16.0%, 16.5%, and 17.0%.

[0033] The present application will be further described below with reference to embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0034] like Figure 1 As shown, the experiment employed a single-factor experimental design, selecting 1440 Hy-Line Brown laying hens at 72 weeks of age with similar body weight and egg production rate. These hens were randomly divided into 5 treatment groups (including 3 example control groups and 2 comparative control groups), with 8 replicates per treatment and 36 hens per replicate. A one-week pre-feeding period was established before the experiment, during which all groups were fed a basal diet free of astaxanthin and canthaxanthin. The experimental period was 6 weeks.

[0035] I. Experimental Materials Astaxanthin fermented with Rhodopsinia rubra (effective astaxanthin content 1.5%) was purchased from Inner Mongolia Jinhe Biotechnology Co., Ltd.

[0036] Cantharidin (10% Tianlihong) is a commercially available product.

[0037] The experimental animals were Hy-Line Brown hens aged 72 weeks with similar weight and egg production rate, which were in the late stage of peak egg production and belonged to late-stage laying hens.

[0038] The egg yolk antioxidant performance test kit (total antioxidant capacity T-AOC, A015-2-1; total superoxide dismutase T-SOD, A001-3-2; DPPH free radical scavenging rate, A153-1-1; malondialdehyde (MDA), A003-1-2) was purchased from Nanjing Jiancheng Biotechnology Institute.

[0039] Electronic tongue (TS-5000Z), INSENT Corporation, Japan.

[0040] Electronic nose (cNose 18), Shanghai Baosheng Industrial Development Co., Ltd.

[0041] Multifunctional enzyme-linked immunosorbent assay (ELISA) instrument (Spectra Max M2e), Molecular Devices, USA.

[0042] Automated biochemical analyzer (HITEC 7100), Hitachi, Japan.

[0043] II. Auxiliary Material Formula The supplementary feed used in this application is a corn-soybean meal type basal diet, fed in powder form, and formulated with reference to the Chinese chicken feeding standards. The composition and nutritional level of the supplementary feed are shown in Table 1.

[0044] Table 1. Composition and Nutritional Level of Excipients Note: Each kilogram of premixed feed provides 2500 IU of vitamin A1, 2500 IU of vitamin D3, 30 mg of vitamin E, 8 mg of copper, 75 mg of zinc, 80 mg of iron, 100 mg of manganese, and 0.15 mg of selenium. Each kilogram of compound enzyme preparation contains 4000 IU of xylanase, 500 IU of phytase, 400 IU of β-mannanase, and 200 IU of β-glucanase.

[0045] III. Experimental Design and Implementation The experiment employed a single-factor experimental design, selecting 1440 72-week-old Hy-Line Brown laying hens, who were randomly divided into 5 treatment groups (including 3 example control groups and 2 comparative control groups), with 8 replicates per treatment and 36 hens per replicate. A one-week pre-feeding period was established before the start of the experiment, during which all groups were fed a supplementary diet (basal diet) without astaxanthin and canthaxanthin. The experimental period lasted for 6 weeks.

[0046] It should be noted that the labels of the processing groups in the accompanying drawings of this application are in abbreviated form, and their correspondence with the embodiments and comparative examples in this specification is as follows: CON response ratio 1 (negative control group, fed supplementary feed only); PC response ratio 2 (positive control group, with 70g / t cantharidin added to the excipients); Example 1 corresponding to ASTA500 (500 g / t of red phloxetine yeast fermented astaxanthin product added to excipients). Example 2 corresponding to ASTA1000 (1000 g / t of red phaf yeast fermented astaxanthin product added to excipients). Example 3 of ASTA2000 (2000 g / t of red phaf yeast fermented astaxanthin product added to excipients).

[0047] Example 1: Astaxanthin fermented with Pharbitis rubra yeast (1.5% effective astaxanthin content) was mixed evenly with the supplementary feed (as shown in Table 1) at an addition rate of 500 g / t to prepare a feed composition. This feed composition was fed to 72-week-old Hy-Line Brown laying hens. They were fed once daily at 7:00 AM with free access to feed and water for 6 consecutive weeks. The temperature was maintained at 22-27℃, relative humidity at 60%, and light duration at 18 hours / day.

[0048] Example 2: The difference from Example 1 is that the amount of astaxanthin product fermented with red phaf yeast added is 1000 g / t. The remaining steps are exactly the same as in Example 1.

[0049] Example 3: The difference from Example 1 is that the amount of astaxanthin product fermented with red phaf yeast added is 2000 g / t. The remaining steps are exactly the same as in Example 1.

[0050] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses excipients, does not add red phaf yeast to ferment the astaxanthin product, and does not add any coloring agents. The remaining steps are exactly the same as in Example 1.

[0051] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the astaxanthin product fermented with red phaf yeast is not added; instead, 70 g / t of canthaxanthin (10% Tianlihong) is added to the excipients. The remaining steps are exactly the same as in Example 1.

[0052] This comparative example is used to compare the differences in reproductive safety between synthetic carotenoid colorants and natural red phaf yeast-fermented astaxanthin.

[0053] Feeding and management: The laying hens were housed in fully enclosed stacked cages, with 4 hens per cage. Each replicate consisted of 9 cages arranged in three layers (top, middle, and bottom) (3 cages per layer). During the trial, eggs were collected and weighed daily at 2:00 PM, and feed consumption was tallied weekly. Immunization was carried out according to standard management procedures.

[0054] IV. Performance Testing Methods 4.1 Production performance: The number of eggs laid and the total egg weight are recorded daily on a repeating basis. The feed consumption is calculated weekly, and the egg production rate, average egg weight, average daily feed intake and feed conversion ratio are calculated.

[0055] 4.2 Follicle development: After the experiment, 8 laying hens were randomly selected from each group for necropsy, ovarian weight was measured, and the number of large yellow follicles (>9mm), small yellow follicles (6-8mm), large white follicles (2-5mm) and total number of follicles (>2mm) were counted. The diameter of the 5 largest grade follicles (F1-F5) was measured.

[0056] 4.3 Egg Quality: A multi-functional egg quality analyzer was used to determine routine indicators such as eggshell color, shell strength, yolk color, albumen height, Haugh unit, and shell thickness. For stored egg quality testing, the eggs were stored at 38-41℃ for 7 days, and the above measurements were repeated.

[0057] 4.4 Antioxidant Properties: The activities of T-AOC and T-SOD, DPPH free radical scavenging rate, and MDA content in egg yolk and serum were determined using a kit from Nanjing Jiancheng Bioengineering Institute. The same method was used to determine T-AOC, T-SOD, and MDA in liver and ovarian tissues. Tissue Sample Pretreatment: An appropriate amount of tissue sample was weighed, 10 times its volume of physiological saline was added, and the sample was ground at low temperature. After centrifugation at 12000 r / min for 5 min at 4℃, the supernatant was collected, and the total protein content of the tissue homogenate was determined. OD values ​​were detected using a multi-functional microplate reader.

[0058] 4.5 Flavor Determination: An electronic tongue was used to determine the basic taste indicators of egg yolk and egg white; an electronic nose was used to determine the sensor response values ​​of 28 volatile substances in egg yolk and egg white. Electronic nose parameter settings: carrier gas flow rate 300 L / min, washing time 120 s, sample injection time 90 s, each sample was measured twice. For electronic tongue detection, approximately 40 g of pulverized egg white and yolk samples were taken, added to 160 mL of pure water, ultrasonically broken up, centrifuged at 5000 rpm for 10 min, and 100 mL of supernatant was collected for testing. Each sample underwent four cycles of testing, and the data from the last three cycles were analyzed.

[0059] 4.6 Astaxanthin content in egg yolks: Egg yolks were separated in replicates, mixed, and the amount of astaxanthin deposited in the yolks was determined by high performance liquid chromatography (HPLC).

[0060] 4.7 Data Analysis: One-way ANOVA was performed using SPSS 24.0 software. Duncan's multiple comparison test was used when differences between groups were significant. P < 0.05 was considered statistically significant. Results are expressed as mean ± standard deviation. Radar charts and principal component analysis plots of the electronic nose and electronic tongue data were generated using OriginPro 2024 software.

[0061] V. Experimental Results and Analysis 5.1 Effects on ovarian and follicular development like Figure 1 As shown in the figure (CON, PC, ASTA500, ASTA1000, and ASTA2000 correspond to Comparative Example 1, Comparative Example 2, Example 1, Example 2, and Example 3, respectively, and the same applies below), compared with Comparative Example 1, Examples 2 and 3 can increase the ovarian weight of laying hens, while Example 1 has no significant effect on ovarian weight.

[0062] Table 2 Number of follicles in each group of laying hens Note: Different letters in the same row indicate significant differences (P<0.05), the same applies to the table below.

[0063] Table 3. Diameter (mm) of the top 5 graded follicles in each group As shown in Tables 2 and 3, the effect of astaxanthin fermented with Pharbitis rubra on follicle development exhibits differential characteristics related to the amount added.

[0064] It is understandable that the decline in ovarian function in laying hens during the later stages of egg production is essentially due to oxidative stress leading to follicular cell damage and apoptosis. Astaxanthin, as a potent antioxidant, can protect the normal development of follicular cells by scavenging free radicals in ovarian tissue and reducing lipid peroxidation damage.

[0065] When the amount of astaxanthin fermented with Phaeformis was added in the range of 500-1000 g / t, Example 1 mainly worked by increasing the reserve of large white follicles, while Example 2 promoted the growth and development of existing graded follicles. When the amount added was further increased to the range of 1000-2000 g / t, the performance of Example 3 was similar to that of Example 1, with the number of large white follicles increasing by 49% and the total number of follicles increasing by 39%.

[0066] It should be noted that although Comparative Example 2 also showed some improvement in follicle indicators, its ovarian T-SOD was significantly reduced to 87.48±3.80U / mgprot (see Table 5 below), while the ovarian T-SOD of all example groups remained within the normal range of 92.99-95.38U / mgprot.

[0067] In traditional methods, cantharidin is often used as an egg yolk coloring agent, but the experimental results of this application show that it poses a risk of oxidative stress damage to the reproductive system of laying hens in the late laying period. However, the astaxanthin fermentation method using Pharrellis redis fermentation adopted in this application improves follicle development while protecting the antioxidant defense capacity of the ovary. This is because the dextrorotatory conformation (3R-3'R) of astaxanthin has free radical scavenging activity, and the polysaccharides and nucleotides in the yeast matrix have a synergistic effect on antioxidant function.

[0068] 5.2 Effects on endogenous antioxidant quality of eggs The results of the antioxidant properties of egg yolks and serum in each group are shown in Table 4, and the results of the antioxidant properties of liver and ovarian tissues in each group are shown in Table 5.

[0069] Table 4 Antioxidant properties of serum and egg yolk in each group As shown in Table 4, compared with Comparative Example 1, adding red phaf yeast-fermented astaxanthin to the diet significantly increased the T-AOC of egg yolks. It should be noted that the T-AOC of egg yolks in Example 1 reached 0.56 ± 0.14 μM / prot, showing no significant difference compared with Examples 2 and 3.

[0070] This indicates that an addition of 500g / t has reached the saturation threshold of the antioxidant capacity of egg yolks, and further increasing the addition amount will not further improve the antioxidant effect.

[0071] Furthermore, compared with Comparative Example 2, it can be seen that although cantharidin can increase the serum DPPH free radical scavenging rate, its egg yolk T-AOC is not significantly different from that of Comparative Example 1, indicating that the antioxidant activity of cantharidin is not effectively transferred to the egg yolk.

[0072] Table 5 Antioxidant properties of liver and ovarian tissues in each group It is understandable that Example 2 significantly reduced liver MDA levels, indicating that astaxanthin exerts its antioxidant protective effect primarily in the liver during its metabolism in laying hens. The liver is the core organ for the synthesis of yolk precursor proteins and lipids in laying hens. Reducing the level of oxidative stress in the liver is beneficial for the normal synthesis and secretion of yolk precursor substances, thereby indirectly promoting follicle development and maintaining egg quality.

[0073] Furthermore, there were no significant differences in liver T-AOC, liver T-SOD, ovarian MDA, and ovarian T-AOC among the groups, indicating that astaxanthin optimizes redox balance within the normal physiological range rather than causing an abnormal antioxidant enzyme response.

[0074] Notably, the ovarian T-SOD in Comparative Example 2 was significantly lower than that in Comparative Example 1 and all other example groups, confirming the risk of oxidative stress damage to the ovaries caused by cantharidin.

[0075] 5.3 Effects on the flavor of egg yolk and egg white The results of electronic tongue assays revealed the selective improvement effect of astaxanthin on the flavor of egg yolks and egg whites.

[0076] Table 6 Flavor profiles of egg yolks and egg whites using electronic tongues in each group. Comparing Examples 1 and 2 with Comparative Example 1, it can be seen that adding 500 g / t and 1000 g / t of red phaf yeast-fermented astaxanthin reduced the bitterness and astringency of egg yolks, while significantly improving umami and saltiness. Example 1 showed the largest reduction in bitterness, decreasing from 2.24 to 0.60. Although Example 3 also significantly reduced bitterness and astringency, the improvement in umami and saltiness was not significant, suggesting that excessively high addition amounts may lead to diminishing marginal returns in flavor improvement.

[0077] Furthermore, as shown in Table 6 regarding the protein data, the bitterness of the protein in each example group was significantly reduced compared to Comparative Example 1, but the umami and saltiness of the protein were significantly reduced compared to Comparative Example 1. This indicates that astaxanthin has different effects on the flavor regulation of egg yolk and egg white: in egg yolk, it mainly manifests as "reducing bitterness and enhancing umami", while in egg white, it mainly manifests as "reducing bitterness and astringency".

[0078] like Figure 3 As shown, electronic nose analysis revealed the differential regulation of volatile flavor compounds in egg yolk and egg white by astaxanthin. Example 3 significantly increased the response values ​​of 22 out of 28 sensors in the egg yolk, mainly responding to aldehydes, ketones, alkanes, alcohols, nitrogenous compounds, sulfides, volatile organic compounds, and aromatics, indicating an increased abundance of volatile flavor compounds in the egg yolk. In contrast, each example group significantly decreased the response values ​​of 18-19 sensors in the egg white, indicating a reduction in the content of undesirable volatile compounds such as sulfides and nitrogenous compounds in the egg white.

[0079] It is understandable that this differentiated effect of "enhancing the flavor of the yolk and removing the fishy smell from the egg white" may be attributed to the fact that astaxanthin is mainly deposited in the lipid layer of the yolk. Its antioxidant effect inhibits the abnormal oxidation of the yolk lipids and protects the beneficial flavor precursors. Meanwhile, the content of undesirable flavor substances in the egg white is reduced under the systemic antioxidant effect of astaxanthin.

[0080] 5.4 Effect on the deposition of astaxanthin in egg yolk Table 7. Astaxanthin deposition in egg yolks of each group Table 7 shows that adding *Phaverus rubra* fermented astaxanthin significantly increased the deposition of astaxanthin in egg yolks. It should be noted that there was no significant difference in deposition among Examples 1, 2, and 3, indicating that 500 g / t reached the deposition plateau for astaxanthin in egg yolks. If calculated based on an effective astaxanthin content of 1.5%, the effective astaxanthin addition at 500 g / t is only 7.5 mg / kg. Further increasing the addition will increase feed costs without further improving deposition. This deposition saturation effect is consistent with the saturation threshold of T-AOC in egg yolks, verifying that 500 g / t is the economically optimal addition for improving egg yolk quality.

[0081] 5.5 Impact on conventional egg quality Table 8 Egg quality indicators for each group It is understandable that astaxanthin fermented with *Phaefflera heliotropium* significantly improves egg yolk color scores in a dose-dependent manner. However, its coloring effect is lower than that of the canthaxanthin group in Comparative Example 2, because astaxanthin and canthaxanthin have different coloring efficiencies. The core objective of this application is not egg yolk coloring, but rather the improvement of endogenous antioxidant quality and follicle development; the natural improvement in egg yolk color is an additional effect. Other conventional egg quality indicators showed no significant differences among the groups, indicating that the addition of *Phaefflera heliotropium* fermented astaxanthin does not adversely affect the basic physicochemical quality of eggs.

[0082] 5.6 Impact on high-temperature storage stability like Figure 2 As shown, after eggs were stored at a high temperature of 38-41℃ for 7 days, the proportion of AA and A grade eggs in each example group increased compared with Comparative Example 1, while the proportion of C grade eggs decreased.

[0083] In existing technologies, extending the shelf life of eggs usually relies on external methods, which are costly and do not alter the intrinsic quality of the eggs. This application achieves "endogenous preservation" of egg quality by establishing an antioxidant defense line in the laying hen and transferring it to the yolk—astaxanthin is deposited in the lipid layer of the yolk, continuously inhibiting lipid peroxidation and protein denaturation during storage, thereby improving the storage stability of eggs from the source.

[0084] 5.7 Impact on Production Performance Table 9. Laying performance of each group of hens (weeks 1-6) It should be noted that Examples 1 and 2, while significantly reducing average daily feed intake, did not have a significant negative impact on egg weight and laying rate, and the feed conversion ratio remained at a level comparable to that of Comparative Example 1. This indicates that within the range of 500-1000 g / t of astaxanthin fermented with Pharrellis rubrum, laying hens can maintain laying performance under reduced feed intake conditions, which may be related to the fact that astaxanthin improves the efficiency of nutrient digestion and absorption.

[0085] 5.8 Economic Benefit Analysis Table 10 Comparison of Economic Benefits of Each Group Note: Astaxanthin is priced at 130 yuan / kg, and canthaxanthin is priced at 205 yuan / kg.

[0086] Comparing Example 1 and Comparative Example 1, it can be seen that adding 500g / t of red Pharf yeast-fermented astaxanthin resulted in a profit increase of 195.37 yuan / month / 10,000 birds with only an increase of about 6.55 yuan / ton in feed price. Comparative Example 2 and Example 3 both led to a decrease in profit. Considering both functional effects and economic benefits, Example 1 is the economically optimal solution for improving the antioxidant quality of egg yolks and astaxanthin deposition, while Example 2 is the functionally optimal solution for improving follicle development.

[0087] Comparing Comparative Example 1 with Examples 1-3, it can be seen that the addition of astaxanthin fermented with Pharbitis rubescens significantly improved several indicators of laying hens in the later stages of egg production.

[0088] Comparing Comparative Example 1 with Example 1, it can be seen that even at the lowest addition level, the T-AOC of egg yolk increased from 0.32 to 0.56 μM / prot, an increase of 75%; at the same time, the bitterness of egg yolk decreased by 73%, the umami flavor increased by 15%, the number of large white follicles increased by 57%, and the total number of follicles increased by 29%. This indicates that when the addition level is in the lower range of 500-1000 g / t, 500 g / t is sufficient to produce significant effects in three dimensions: antioxidant, flavor, and follicle reserve.

[0089] Comparing Comparative Example 1 and Example 2, it can be seen that when the addition amount is 1000g / t, it shows the best performance in terms of increasing the diameter of graded follicles, while the liver MDA is significantly reduced. This indicates that the antioxidant protection of astaxanthin in the body extends from the liver to the ovary, supporting the development of graded follicles.

[0090] A horizontal comparison between Examples 1-3 shows that when the amount of astaxanthin added increased from 500 g / t to 2000 g / t, the astaxanthin deposition in egg yolks and T-AOC did not significantly increase further, confirming that 500 g / t is the saturation threshold for deposition and antioxidant effects. However, in terms of follicle development, different addition amounts showed different functional focuses: 500 g / t and 2000 g / t focused on supplementing the follicle pool, while 1000 g / t focused on the individual development of graded follicles. Therefore, for different functional needs, farmers can flexibly choose the addition amount within the range of 500-1000 g / t or 1000-2000 g / t.

[0091] Comparing Comparative Example 2 with Examples 1-3, it can be seen that although cantharidin has the strongest effect in egg yolk coloring, its ovarian T-SOD is significantly reduced, indicating that cantharidin poses a risk of oxidative stress damage to the reproductive system of laying hens in the late laying period.

[0092] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. The same components and parameters are represented by the same terms. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. Application of red phaf yeast fermented astaxanthin in the preparation of a composition for improving follicle development and egg quality in late-laying hens.

2. The application according to claim 1, characterized in that, The effective content of astaxanthin in the astaxanthin fermented by the red phaf yeast is 1-2%.

3. The application according to claim 1, characterized in that, The amount of astaxanthin fermented by the red phaf yeast added to the feed is 500-2000 g / t.

4. The application according to claim 3, characterized in that, The amount of astaxanthin fermented with the red phaf yeast added to the feed is 500-1000 g / t; or The amount of astaxanthin fermented by the red phaf yeast added to the feed is 1000-2000g / t.

5. The application according to claim 3, characterized in that, The continuous feeding cycle for astaxanthin fermented by the red phaf yeast is no less than three weeks.

6. A composition for improving follicle development and egg quality in laying hens during the later stages of egg production, characterized in that, Including astaxanthin fermented with red phloxetine and excipients.

7. A functional egg, characterized in that, The functional eggs produced by feeding the composition of claim 6 have a T-AOC ≥ 0.50 μM / prot and an astaxanthin deposition in the yolk ≥ 70 μg / g.

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