Method for preparing laying hen feed additive by fermenting tea-seed oil deodorized distillate

Abstract

The invention discloses a method for preparing a laying hen feed additive by fermenting tea-seed oil deodorized distillate, which is characterized in that the tea-seed oil deodorized distillate is used as a raw material, and efficient utilization of all components of resources is achieved by coupling yeast-paecilomyces cicadae two-step sequential fermentation and endogenous mevalonic acid (MVA) pathway cascade regulation and control technology. The final product is rich in ergosterol, vitamin E and probiotics, can improve the laying rate of laying hens and enhance the eggshell strength after being applied to laying hen breeding, realizes zero wastewater discharge in the process, and achieves the dual goals of resource utilization of grease byproducts and accurate matching of laying hen nutrition.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a method for the resource-based transformation of oil refining byproducts, and particularly to a method for preparing a functional feed additive for laying hens rich in ergosterol, natural vitamin E and probiotic protein by using tea seed oil deodorized distillate as raw material and through a two-step sequential microbial fermentation and metabolic regulation. Background Technology

[0002] Tea seed oil, a high-quality woody edible oil with unique advantages in my country, produces tea seed oil deodorizer distillate (TSODD) during the refining process. This byproduct is rich in phytosterols (15-25%), tocopherols (i.e., vitamin E, 5-12%), free fatty acids (40-60%), and glycerides, among other valuable components, making it an important biomass resource.

[0003] Currently, at the industrial level, the main method for utilizing this byproduct is the saponification-extraction-crystallization chemical method to extract phytosterols. However, this method has significant drawbacks: the use of strong alkali saponification leads to an oxidation loss rate of 30-40% for tocopherols; phytosterols cross-linked with fatty acid glycerides are difficult to completely separate, with a recovery rate of less than 60%; the use of high-concentration alkaline solutions and organic solvents not only increases the cost of waste treatment and creates environmental pressure, but also only allows for the single utilization of sterol components, while other components are discarded, failing to achieve the resource utilization of all components.

[0004] Ergosterol, a precursor to vitamin D2, is converted into vitamin D2 upon ultraviolet irradiation, significantly promoting calcium and phosphorus metabolism in laying hens and improving eggshell quality. Furthermore, its absorption rate in the intestine is 2-3 times higher than that of phytosterols, and it can also activate macrophages, enhancing the disease resistance of poultry. It is a key functional component in the nutrition of laying hens. Traditionally, ergosterol is mainly derived from yeast powder, which presents problems such as high cost and animal-borne disease risks. Therefore, there is an urgent need to develop plant-derived conversion and preparation technologies.

[0005] Currently, most studies on the fermentation of deodorized oil distillate use a single yeast strain, which has significant limitations. Specifically, the product spectrum is relatively limited, mainly synthesizing sitosterol and campesterol, with ergosterol conversion rate below 1%; substrate utilization is insufficient, with fatty acid conversion rate only 70-80%, and residual oil adversely affecting subsequent processing and application; the fermentation products are not precisely tailored to the nutritional needs of laying hens, lacking systematic formulation design, and have relatively limited functions, failing to fully realize synergistic nutritional effects.

[0006] Therefore, developing a highly efficient, green technology that enables the full utilization of all components of deodorized tea seed oil distillate, achieving the targeted and efficient synthesis of ergosterol, and simultaneously enriching various essential nutrients for laying hens, is a pressing technical challenge that needs to be addressed in the field of oil by-product resource utilization and laying hen feed additives. Summary of the Invention

[0007] (I) Purpose of the Invention

[0008] This invention aims to overcome the shortcomings of existing technologies and provide a method for fermenting tea seed oil deodorized distillate into a feed additive for laying hens. Specific objectives include:

[0009] To achieve efficient utilization of all components of deodorized tea seed oil distillate, solving the problems of low utilization rate and serious pollution caused by traditional chemical methods;

[0010] Through a two-step sequential fermentation and metabolic regulation, phytosterols are directionally converted into high-value-added ergosterol, resulting in an ergosterol content of ≥0.5g / kg (dry basis) in the product.

[0011] It simultaneously enriches natural vitamin E, probiotic protein, antimicrobial peptides and other functional ingredients to achieve synergistic nutritional enhancement;

[0012] Based on the nutritional needs of laying hens during the egg-laying period, calcium, phosphorus, and other carriers are precisely compounded to prepare functional feed additives that are suitable for the physiological characteristics of laying hens.

[0013] Simplify the process, reduce costs, achieve green and environmentally friendly practices, and eliminate wastewater discharge.

[0014] (II) Technical Solution

[0015] To achieve the objectives of this invention, the following technical solution is adopted:

[0016] A method for preparing a feed additive for laying hens by fermentation of deodorized tea seed oil distillate, characterized by comprising the following steps:

[0017] (1) Raw material pretreatment: The deodorized distillate of tea seed oil is heated to 60-65℃ and centrifuged at a speed of 6000-8000 rpm to remove impurities and obtain a clear oily liquid.

[0018] (2) First step: Yeast liquid fermentation: Using the pretreated deodorized distillate as the main carbon source, a fermentation medium was prepared, and Candida utilis or Saccharomyces cerevisiae seed liquid was inoculated. Fermentation was carried out at 30-37℃, with an aeration rate of 0.5-0.8 volumes / (volume·min) and a stirring speed of 300-500 rpm for 36-48 hours until the free fatty acid content was ≤8 g / L and the glycerol ester hydrolysis rate was ≥75%.

[0019] (3) Second step: Solid-state fermentation of *Penicillium cicadae*: The fermentation product from the first step is mixed with wheat bran, rice husks, soybean meal, etc., to prepare a solid substrate. *Penicillium cicadae* spore suspension is then inoculated into the substrate, and fermentation is carried out for 72-96 hours through a three-stage cascade regulation method: mycelial proliferation phase - metabolic flux regulation phase - product stabilization phase. The cascade regulation measures include: nitrogen restriction treatment after 24 hours of fermentation; controlling the iron citrate content in the substrate to 0.01%-0.03% to achieve iron restriction; performing dissolved oxygen pulse operation every 12 hours; lowering the temperature to 24-26℃ after 48 hours of fermentation; and adjusting the pH to 5.0-5.5 after 36 and 60 hours of fermentation.

[0020] (4) Fermentation product processing: The solid fermentation product is placed at 45-50℃ for vacuum drying until the moisture content is ≤8%. Then it is subjected to ultra-fine pulverization and passed through a 60-80 mesh sieve to obtain the fermentation core material.

[0021] (5) Compound preparation: According to the weight percentage, 40%-50% of fermentation core material, 25%-30% of stone powder, 10%-15% of dicalcium phosphate, 8%-12% of zeolite powder, 2%-4% of yeast cell wall polysaccharide and 0.02%-0.05% of ethoxyquinoline are uniformly mixed to obtain the laying hen feed additive.

[0022] The *Candida utilis* strain used in step (2) is *Candida utilis* AS2.1180, and the *Saccharomyces cerevisiae* strain is *Saccharomyces cerevisiae* CICC 1001. The fermentation medium formula is as follows: 100-150 g / L deodorized distillate, 5-8 g / L (NH4)2SO4, 0.5-1.0 g / L MgSO4·7H2O, 2-3 g / L KH2PO4, and an initial pH of 5.5-6.0. The inoculum size of the seed culture is 8-12% (v / v), and the concentration of the seed culture cells is not less than 1×10⁻⁶. 8 CFU / mL.

[0023] The Paecilomycescicadae in step (3) is CGMCC 3.3790; the solid substrate formula (by dry weight percentage) is: 45-55% yeast fermentation broth, 20-30% wheat bran, 10-15% rice husk, 8-12% soybean meal, 0.01-0.03% ferric citrate, 0.5-1.0% KH2PO4, 0.1-0.2% MgSO4·7H2O, and the substrate moisture content is 58-62%.

[0024] The concentration of the spore suspension in step (3) is 1×10⁻⁶. 8 -5×10 8The inoculum concentration is 5-8% (v / w), and a solution containing 0.05% Tween-80 is sprayed during inoculation. The temperature during the mycelial proliferation phase is 25-28℃, and the temperature during the product stabilization phase is 26-28℃.

[0025] In step (2), the extracellular lipase activity during yeast fermentation is not less than 50 U / mL, and sterol esters are converted into free phytosterols through enzymatic hydrolysis.

[0026] The cascade regulation in step (3) does not require the addition of exogenous mevalonic acid. By regulating the process parameters, the activity of HMG-CoA reductase is increased by 2.3-3.1 times, and the transcriptional level of mevalonic acid kinase is upregulated by 4.5 times.

[0027] At the end of fermentation in step (3), the ergosterol content shall not be less than 0.50 mg / g (dry basis), the total sterol retention rate shall not be less than 90%, the mycelial biomass shall not be less than 0.40 g / g dry basis, and the free fatty acid residue shall not be higher than 2.5 g / kg.

[0028] The compounded product in step (5) meets the following indicators: vitamin D not less than 1600 IU / kg, vitamin E not less than 5 IU / kg, crude protein not less than 16.5%, calcium content 3.5%, phosphorus content 0.6%, and probiotic live bacteria count not less than 2×10⁻⁶. 9 CFU / g.

[0029] In step (4), the fermentation product is directly dried and pulverized without separation and purification. After the mycelium of *Penicillium cicadae* breaks naturally, the intracellular ergosterol release rate is greater than 85%.

[0030] The application of egg-laying hen feed additives in egg-laying hen farming is characterized by the following: the amount of the additive added to the daily diet of egg-laying hens is 0.2-0.5%, which can improve egg production rate, improve eggshell quality, increase the vitamin D3 content of egg yolks, and regulate intestinal flora.

[0031] (III) Beneficial Effects

[0032] Compared with the prior art, the present invention has the following significant advantages:

[0033] 1. High-efficiency utilization of resources: It realizes the effective utilization of all components of tea seed oil deodorized distillate, with the conversion rate of free fatty acids in the range of 85-90% and the retention rate of sterols not less than 90%. It solves the problem of traditional methods that only utilize sterols and waste resources, and improves the resource utilization value of oil by-products.

[0034] 2. Complex Product Functions: By simultaneously enriching ergosterol, natural vitamin E, probiotic protein, antimicrobial peptides, and yeast cell wall polysaccharides through two-step fermentation, a four-fold synergistic system is constructed. Compared with single-component additives, the bioavailability is increased by 2-3 times, which can precisely meet the nutritional needs of laying hens during the egg-laying period.

[0035] 3. Metabolic Regulation Innovation: The first non-GMO strategy of regulating the endogenous MVA pathway through a cascade of fermentation process parameters, using a combination of nitrogen restriction, iron restriction, and dissolved oxygen pulse regulation, increases the ergosterol synthesis throughput by 30-40%, breaking through the technical bottleneck of low ergosterol conversion rate in traditional fermentation.

[0036] 4. Green and energy-saving process: The process coupling design of "direct use of yeast fermentation broth as solid fermentation substrate" eliminates the need for sterilization and separation purification operations, and eliminates high-energy-consuming units such as filter cake-oil layer separation and high-pressure homogenization. Post-processing energy consumption is reduced by 55-65%, equipment investment is reduced by more than 50%, and there is no wastewater discharge, which meets green standards.

[0037] 5. Significant application effects: Applying the product to egg-laying hen farming can increase egg production rate by 5.8-7.5%, improve eggshell strength by 12-16%, reduce egg breakage rate by 45%, increase the content of 25-hydroxyvitamin D3 in egg yolk by 3.5-4.0 times, increase the number of cecal lactic acid bacteria by 1.8 orders of magnitude, and reduce the overall cost by 60-65% compared to chemical purification methods, thus achieving both economic and social benefits.

[0038] 6. Reliable safety performance: No chemical reagents are added during the fermentation process. The products are natural microbial metabolites and plant-derived components, which avoids the disease risks associated with animal-derived ergosterol. The product meets feed safety standards and is highly safe to use. Detailed Implementation

[0039] Example 1: The core technical solution adopted in this invention is a cascade regulation strategy of yeast-Penicillium cicadae two-step sequential fermentation coupled with endogenous mevalonic acid (MVA) pathway.

[0040] A method for preparing a feed additive for laying hens by fermentation of deodorized tea seed oil distillate, the specific steps of which are as follows:

[0041] Raw material pretreatment: Select tea seed oil deodorized distillate with an acid value in the range of 80 mg KOH / g and a sterol content of not less than 18%, heat it to 60℃, and centrifuge it at a speed of 6000 r / min to remove mechanical impurities and polymer pigments, and obtain a clear oily liquid for later use.

[0042] Step 1: Yeast liquid fermentation (fatty acid degradation and sterol release stage)

[0043] (1) Strain screening

[0044] Candida utilis AS2.1180 or Saccharomyces cerevisiae CICC1001 were selected. These yeasts have the ability to efficiently degrade fatty acids and glycerides, and can also secrete extracellular lipases.

[0045] (2) Seed liquid preparation

[0046] The bacterial strain was inoculated from a slant onto YPD liquid medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and activated by incubation at 28°C and 200 rpm for 24 h. Then, acclimatization medium containing 5% (w / w) deodorized distillate was added at a 10% (v / v) inoculation rate and incubated at 37°C for 12 h until the bacterial concentration reached ≥1×10⁻⁶. 8 CFU / mL.

[0047] (3) Fermentation operation

[0048] The fermentation medium was prepared with the following formula: 100 g / L deodorized distillate, 5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, and 3 g / L KH2PO4, with an initial pH of 5.5-6.0. Seed culture was inoculated at an inoculum rate of 8% (v / v) and fermented for 36 h at 30℃, aeration rate of 0.5 vvm, stirring speed of 500 rpm, and dissolved oxygen ≥20%. The final fermentation endpoint was controlled at pH 4.5, free fatty acid content ≤8 g / L (conversion rate 85-90%), and glycerol ester hydrolysis rate ≥75%.

[0049] (4) Key technologies

[0050] During the metabolism of fatty acids, yeast produces extracellular lipases (enzyme activity ≥50U / mL). These enzymes can catalyze the hydrolysis of sterol esters, converting bound sterols into free sterols, thereby providing substrates for the second step of ergosterol synthesis.

[0051] Step 2: Solid-state fermentation of *Penicillium cicadae* (ergosterol synthesis and metabolic regulation stage)

[0052] (1) Strain screening

[0053] Paecilomyces cescicadae CGMCC 3.3790 was selected. This strain has the ability to synthesize ergosterol from phytosterols naturally, and the endogenous mevalonic acid (MVA) pathway can be activated through process regulation.

[0054] (2) Solid matrix preparation

[0055] Using the first-step yeast fermentation product as a functional matrix, the residual yeast cell wall polysaccharides, polypeptides, and trace elements can activate the secondary metabolism of *Penicillium cicadae*. The matrix formula (by dry weight percentage) is as follows: 45% yeast fermentation broth, 30% wheat bran, 10% rice husk, 8% soybean meal, 0.01% ferric citrate, 1.0% potassium dihydrogen phosphate (KH2PO4), and 0.1% magnesium sulfate heptahydrate (MgSO4·7H2O), with the moisture content adjusted to 58%.

[0056] (3) Vaccination procedure

[0057] The preparation concentration is 1×10 8 A spore suspension of *Penicillium cicadae* at 8% (v / w) was inoculated into a solid substrate. A solution containing 0.05% Tween-80 was sprayed at inoculation to promote spore germination and substrate penetration.

[0058] (4) Cascade-regulated fermentation

[0059] During the mycelial proliferation period (0-24h), the temperature is controlled at 25℃, natural ventilation is used, the temperature at the center of the substrate layer is controlled not to exceed 30℃, and the pH is naturally maintained at 5.8 to promote the accumulation of mycelial biomass.

[0060] Metabolic flux regulation period (24-72h): At 24h, the substrate was turned over and nitrogen supplementation was stopped (i.e., nitrogen restriction regulation was implemented); iron restriction regulation was achieved by controlling the iron citrate content in the substrate to 0.01%, thereby relieving the transcriptional inhibition of the ERG gene cluster by the FET4 iron transporter; forced ventilation (air volume of 0.3 v / vm) was performed for 30 minutes every 12h to achieve dissolved oxygen pulse and stimulate ERG5 activity; from 48h, the temperature was lowered to 24℃ (i.e., low temperature stress treatment) to upregulate ERG6 expression; citric acid was sprayed at 36h and 60h respectively to instantly lower the surface pH to 5.0 (i.e., pH stress treatment) to activate secondary metabolism.

[0061] Product stabilization period (72-96h): Maintain the temperature at 26℃ and turn the substrate every 24h to prevent ergosterol oxidation. Stop fermentation when the mycelium covers the substrate and the substrate temperature approaches ambient temperature.

[0062] (5) Endpoint Indicators

[0063] The ergosterol content is not less than 0.5 g / kg (dry basis), the total sterol retention rate is not less than 90%, the mycelial biomass is not less than 4% (dry basis), and the free fatty acid residue is not more than 2.5 g / kg.

[0064] The fermentation product processing involves directly transferring the solid fermentation substrate into a vacuum dryer and drying it at 50°C and a vacuum of -0.08 MPa until the moisture content is ≤8%, without the need for separation and purification, thus retaining all active ingredients. The substrate is then passed through an 80-mesh sieve using a shear pulverizer to obtain a uniform fermentation core material. The mycelium of *Penicillium cicadae* naturally breaks down, and the intracellular ergosterol release rate is >85%.

[0065] The feed additives were formulated and prepared according to the nutritional requirements of laying hens in NY / T33-2004 "Chicken Feeding Standards". The following ingredients were formulated by weight percentage: 40% fermented core feed, 30% limestone powder, 10% dicalcium phosphate, 12% zeolite powder, 2% yeast cell wall polysaccharide, and 0.02% ethoxyquinoline. The mixture was stirred in a double helix mixer for 20 minutes, with the coefficient of variation (CV) controlled to be ≤5%. The final product was obtained after quality testing (negative for coliform bacteria and Salmonella).

[0066] Example 2: The core technical solution adopted in this invention is a cascade regulation strategy of yeast-Penicillium cicadae two-step sequential fermentation coupled with endogenous mevalonic acid (MVA) pathway.

[0067] A method for preparing a feed additive for laying hens by fermentation of deodorized tea seed oil distillate, the specific steps of which are as follows:

[0068] Raw material pretreatment: Select tea seed oil deodorized distillate with an acid value in the range of 120 mg KOH / g and a sterol content of not less than 18%, heat it to 65℃, and centrifuge it at a speed of 8000 r / min to remove mechanical impurities and polymer pigments, and obtain a clear oily liquid for later use.

[0069] Step 1: Yeast liquid fermentation (fatty acid degradation and sterol release stage)

[0070] (1) Strain screening

[0071] Candida utilis AS2.1180 or Saccharomyces cerevisiae CICC1001 were selected. These yeasts have the ability to efficiently degrade fatty acids and glycerides, and can also secrete extracellular lipases.

[0072] (2) Seed liquid preparation

[0073] The bacterial strain was inoculated from a slant onto YPD liquid medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and activated by incubation at 37°C and 200 rpm for 18 h. Then, acclimatization medium containing 5% (w / w) deodorized distillate was added at a 10% (v / v) inoculation rate and incubated at 37°C for 16 h until the bacterial concentration reached ≥1×10⁻⁶. 8 CFU / mL.

[0074] (3) Fermentation operation

[0075] The fermentation medium was prepared with the following formula: 150 g / L deodorized distillate, 8 g / L (NH4)2SO4, 1.0 g / L MgSO4·7H2O, and 2 g / L KH2PO4, with an initial pH of 6.0. Seed culture was inoculated at a rate of 12% (v / v) and fermented for 36 h at 30℃, aeration rate of 0.8 vvm, stirring speed of 300-500 rpm, and dissolved oxygen ≥20%. The final fermentation endpoint was controlled at pH 4.5, free fatty acid content ≤8 g / L (conversion rate 85-90%), and glycerol ester hydrolysis rate ≥75%.

[0076] (4) Key technologies

[0077] During the metabolism of fatty acids, yeast produces extracellular lipases (enzyme activity ≥50U / mL). These enzymes can catalyze the hydrolysis of sterol esters, converting bound sterols into free sterols, thereby providing substrates for the second step of ergosterol synthesis.

[0078] Step 2: Solid-state fermentation of *Penicillium cicadae* (ergosterol synthesis and metabolic regulation stage)

[0079] (1) Strain screening

[0080] Paecilomyces cescicadae CGMCC 3.3790 was selected. This strain has the ability to synthesize ergosterol from phytosterols naturally, and the endogenous mevalonic acid (MVA) pathway can be activated through process regulation.

[0081] (2) Solid matrix preparation

[0082] Using the first-step yeast fermentation product as a functional matrix, the residual yeast cell wall polysaccharides, polypeptides, and trace elements can activate the secondary metabolism of *Penicillium ciliata*. The matrix formula (by dry weight percentage) is as follows: 55% yeast fermentation broth, 20% wheat bran, 15% rice husk, 8% soybean meal powder, 0.03% ferric citrate, 1.0% potassium dihydrogen phosphate (KH2PO4), and 0.2% magnesium sulfate heptahydrate (MgSO4·7H2O), with the moisture content adjusted to 62%.

[0083] (3) Vaccination procedure

[0084] The preparation concentration is 5×10 8 A spore suspension of *Penicillium cicadae* at 5% (v / w) was inoculated into a solid substrate. A solution containing 0.05% Tween-80 was sprayed at inoculation to promote spore germination and substrate penetration.

[0085] (4) Cascade-regulated fermentation

[0086] During the mycelial proliferation period (0-24h), the temperature is controlled at 28℃, natural ventilation is used, the temperature at the center of the substrate layer is controlled not to exceed 30℃, and the pH is naturally maintained at 6.2 to promote the accumulation of mycelial biomass.

[0087] Metabolic flux regulation period (24-72h): At 24h, the substrate was turned over and nitrogen supplementation was stopped (i.e., nitrogen restriction regulation was implemented); iron restriction regulation was achieved by controlling the iron citrate content in the substrate to 0.03%, thereby relieving the transcriptional inhibition of the ERG gene cluster by the FET4 iron transporter; forced ventilation (air volume of 0.3 vvm) was performed for 30 minutes every 12h to achieve dissolved oxygen pulse and stimulate ERG5 activity; from 48h, the temperature was lowered to 26℃ (i.e., low temperature stress treatment) to upregulate ERG6 expression; citric acid was sprayed at 36h and 60h respectively to instantly lower the surface pH to 5.5 (i.e., pH stress treatment) to activate secondary metabolism.

[0088] Product stabilization period (72-96h): Maintain the temperature at 28℃ and turn the substrate every 24h to prevent ergosterol oxidation. Stop fermentation when the mycelium covers the substrate and the substrate temperature approaches ambient temperature.

[0089] (5) Endpoint Indicators

[0090] The ergosterol content is not less than 0.5 g / kg (dry basis), the total sterol retention rate is not less than 90%, the mycelial biomass is not less than 4% (dry basis), and the free fatty acid residue is not more than 2.5 g / kg.

[0091] The fermentation product processing involves directly transferring the solid fermentation substrate into a vacuum dryer and drying it at 45℃ and a vacuum of -0.08MPa until the moisture content is ≤8%, without the need for separation and purification, thus retaining all active ingredients. The substrate is then passed through a shear pulverizer and a 60-mesh sieve to obtain a uniform fermentation core material. The mycelium of *Penicillium cicadae* breaks naturally, and the intracellular ergosterol release rate is >85%.

[0092] The feed additive compounding and preparation are based on the nutritional requirements of laying hens in NY / T33-2004 "Chicken Feeding Standards". The compounding is carried out by weight percentage as follows: 50% fermented core feed, 25% limestone powder, 15% dicalcium phosphate, 8% zeolite powder, 4% yeast cell wall polysaccharide, and 0.05% ethoxyquinoline. The mixture is mixed in a double helix mixer for 30 minutes, and the coefficient of variation (CV) is controlled to be ≤5%. The final product is obtained after quality testing (negative for coliform bacteria and Salmonella).

[0093] Example 3: The core technical solution adopted in this invention is a cascade regulation strategy of yeast-Penicillium cicada two-step sequential fermentation coupled with endogenous mevalonic acid (MVA) pathway.

[0094] A method for preparing a feed additive for laying hens by fermentation of deodorized tea seed oil distillate, the specific steps of which are as follows:

[0095] Raw material pretreatment: Select tea seed oil deodorized distillate with an acid value in the range of 100 mg KOH / g and a sterol content of not less than 18%, heat it to 63℃, and centrifuge it at a speed of 7500 r / min to remove mechanical impurities and polymer pigments, and obtain a clear oily liquid for later use.

[0096] Step 1: Yeast liquid fermentation (fatty acid degradation and sterol release stage)

[0097] (1) Strain screening

[0098] Candida utilis AS2.1180 or Saccharomyces cerevisiae CICC1001 were selected. These yeasts have the ability to efficiently degrade fatty acids and glycerides, and can also secrete extracellular lipases.

[0099] (2) Seed liquid preparation

[0100] The bacterial strain was inoculated from a slant onto YPD liquid medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) and activated by incubation at 30°C and 200 rpm for 20 h. Then, an acclimatization medium containing 4% (w / w) deodorized distillate was added at an inoculation rate of 8% (v / v) and incubated at 37°C for 14 h until the bacterial concentration reached ≥1×10⁻⁶. 8 CFU / mL.

[0101] (3) Fermentation operation

[0102] The fermentation medium was prepared with the following formula: 120 g / L deodorized distillate, 7 g / L (NH4)2SO4, 0.8 g / L MgSO4·7H2O, 2.5 g / L KH2PO4, and an initial pH of 6. Seed culture was inoculated at a rate of 10% (v / v) and fermented for 40 h at 35 °C, aeration rate of 0.7 vvm, stirring speed of 400 rpm, and dissolved oxygen ≥20%. The final fermentation endpoint was controlled at pH 4.1, free fatty acid content ≤8 g / L (conversion rate 85-90%), and glycerol ester hydrolysis rate ≥75%.

[0103] (4) Key technologies

[0104] During the metabolism of fatty acids, yeast produces extracellular lipases (enzyme activity ≥50U / mL). These enzymes can catalyze the hydrolysis of sterol esters, converting bound sterols into free sterols, thereby providing substrates for the second step of ergosterol synthesis.

[0105] Step 2: Solid-state fermentation of *Penicillium cicadae* (ergosterol synthesis and metabolic regulation stage)

[0106] (1) Strain screening

[0107] Paecilomyces cescicadae CGMCC 3.3790 was selected. This strain has the ability to synthesize ergosterol from phytosterols naturally, and the endogenous mevalonic acid (MVA) pathway can be activated through process regulation.

[0108] (2) Solid matrix preparation

[0109] Using the first-step yeast fermentation product as a functional matrix, the residual yeast cell wall polysaccharides, polypeptides, and trace elements can activate the secondary metabolism of *Penicillium cicadae*. The matrix formula (by dry weight percentage) is as follows: 50% yeast fermentation broth, 25% wheat bran, 12% rice husk, 10% soybean meal, 0.02% ferric citrate, 0.7% potassium dihydrogen phosphate (KH2PO4), and 0.15% magnesium sulfate heptahydrate (MgSO4·7H2O), with the moisture content adjusted to 60%.

[0110] (3) Vaccination procedure

[0111] The preparation concentration is 3×10 8 A spore suspension of *Penicillium cicadae* at spores / mL was inoculated into a solid substrate at an inoculum rate of 6% (v / w). A solution containing 0.05% Tween-80 was sprayed at inoculation to promote spore germination and substrate penetration.

[0112] (4) Cascade-regulated fermentation

[0113] During the mycelial proliferation period (0-24h), the temperature is controlled at 27℃, natural ventilation is used, the temperature at the center of the substrate layer is controlled not to exceed 30℃, and the pH is naturally maintained at 6.0 to promote the accumulation of mycelial biomass.

[0114] Metabolic flux regulation period (24-72h): At 24h, the substrate was turned over and nitrogen supplementation was stopped (i.e., nitrogen restriction regulation was implemented); iron restriction regulation was achieved by controlling the iron citrate content in the substrate to 0.02%, thereby relieving the transcriptional inhibition of the ERG gene cluster by the FET4 iron transporter; forced ventilation (air volume of 0.3 vvm) was performed for 30 minutes every 12h to achieve dissolved oxygen pulse and stimulate ERG5 activity; from 48h, the temperature was lowered to 25℃ (i.e., low temperature stress treatment) to upregulate ERG6 expression; citric acid was sprayed at 36h and 60h respectively to instantly lower the surface pH to 5.5 (i.e., pH stress treatment) to activate secondary metabolism.

[0115] Product stabilization period (72-96h): Maintain the temperature at 27℃ and turn the substrate every 24h to prevent ergosterol oxidation. Stop fermentation when the mycelium covers the substrate and the substrate temperature approaches ambient temperature.

[0116] (5) Endpoint Indicators

[0117] The ergosterol content is not less than 0.5 g / kg (dry basis), the total sterol retention rate is not less than 90%, the mycelial biomass is not less than 4% (dry basis), and the free fatty acid residue is not more than 2.5 g / kg.

[0118] The solid fermentation substrate was directly transferred to a vacuum dryer and dried to a moisture content of ≤8% at 45℃ and a vacuum of -0.08MPa. No separation or purification was required, and all active ingredients were retained. The substrate was then passed through a shear pulverizer and sieved through a 70-mesh sieve to obtain a uniform fermentation core material. The mycelium of *Penicillium cicadae* naturally broke, and the intracellular ergosterol release rate was >85%.

[0119] The feed additives were formulated and prepared according to the nutritional requirements of laying hens in NY / T33-2004 "Chicken Feeding Standards". The feed additives were formulated by weight percentage as follows: 45% fermented core feed, 27% limestone powder, 12% dicalcium phosphate, 10% zeolite powder, 3% yeast cell wall polysaccharide, and 0.04% ethoxyquinoline. The mixture was mixed in a double helix mixer for 25 minutes, with the coefficient of variation (CV) controlled to be ≤5%. The final product was obtained after quality testing (negative for coliform bacteria and Salmonella).

[0120] Experimental materials and grouping

[0121] experimental animals

[0122] Four hundred and eighty Hy-Line Brown laying hens, aged 48 weeks, of uniform health, with stable egg production (average egg production rate of 78±2%), and weighing between 1.8 and 2.0 kg, were selected. They were randomly divided into four groups of 120 each, with six replicates per group and 20 hens per replicate.

[0123] Experimental Groups

[0124] Test Group Details Table

[0125]

[0126]

[0127] Basal Diet: A basal diet for laying hens was formulated according to NY / T33-2004 "Chicken Feeding Standards," with the following composition: corn 62.5%, soybean meal 22.0%, wheat bran 4.0%, limestone 8.0%, dicalcium phosphate 2.0%, salt 0.3%, and premix 1.2% (containing amino acids, minerals, and basic vitamins, but excluding additional ergosterol and vitamin E). The nutritional levels of the basal diet were: crude protein 16.5%, metabolizable energy 11.2 MJ / kg, calcium 3.5%, and total phosphorus 0.6%.

[0128] Feeding and Management

[0129] The trial lasted for 8 weeks (1 week for the pre-trial period and 7 weeks for the formal trial period), using a three-tiered tiered cage system with 2 chickens per cage and a stocking density of 6 chickens / m². 2 .

[0130] The environmental conditions were set as follows: temperature 18-22℃, relative humidity 55-65%, light cycle of 16h light / 8h darkness, and light intensity of 30 lux.

[0131] The experimental chickens had free access to feed and water, their daily feed intake was recorded, their droppings were cleaned weekly, and disinfection was carried out regularly to ensure a consistent rearing environment.

[0132] Sample collection and testing methods

[0133] Egg production performance monitoring: During the trial period, the number of eggs laid, broken eggs, and deformed eggs were recorded daily for each group, and the daily egg production rate, average egg weight, and breakage rate were calculated. Egg samples (30 eggs per group) were collected weekly for testing eggshell quality and yolk nutritional indicators.

[0134] Eggshell quality testing: Eggshell strength was measured using an eggshell strength tester (Model EFG-0503, Japan); average eggshell thickness (average value of three points: blunt end, middle end, and sharp end) was measured using an eggshell thickness gauge (Model QCT, China); eggshell ultrastructure (density of papillae layer and thickness of palisade layer) was observed using a scanning electron microscope (SEM, SU8010, Japan); and eggshell calcium and phosphorus content was determined using an inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7900, USA).

[0135] Statistical analysis: All data were analyzed using one-way ANOVA, and Duncan's method was used for multiple comparisons. P < 0.05 indicated a significant difference, and P < 0.01 indicated a highly significant difference. Results are expressed as mean ± standard deviation (Mean ± SD).

[0136] Test results and technical effects

[0137] (1) Egg production performance

[0138] Comparison table of egg production performance improvement effects

[0139]

[0140] The results showed that the daily egg production rate of the experimental group (T group) reached 84.3±1.2%, which was significantly higher than the blank control group (CK3 group, 78.5±1.5%) by 7.4% (P<0.01), significantly higher than the single-component control group (CK2 group, 81.2±1.3%) by 3.8% (P<0.05), and significantly higher than the unfermented control group (CK1 group, 77.3±1.6%) by 9.1% (P<0.01). The average egg weight of the experimental group was 62.8±1.1g, which was significantly higher than the CK3 group (60.5±1.0g) by 3.8% (P<0.05); the egg breakage rate was only 1.2±0.3%, which was 45.5% lower than the CK3 group (2.2±0.4%) and 33.3% lower than the CK2 group (1.8±0.3%) (P<0.05). The product of this invention exhibits a synergistic effect from ergosterol (a precursor to vitamin D2), natural vitamin E, probiotics, and yeast cell wall polysaccharides. Vitamin D2, converted from ergosterol, promotes calcium and phosphorus absorption; probiotics regulate intestinal flora balance to improve nutrient utilization; and yeast cell wall polysaccharides enhance the body's immunity. These three components work synergistically to improve the physiological condition of laying hens, and their nutritional synergistic effect is more pronounced than that of single-component additions (CK2 group).

[0141] (2) Eggshell quality

[0142] Eggshell quality improvement effect comparison table

[0143]

[0144] The eggshell strength of the experimental group reached 3.85 ± 0.12 kg / cm². 2 Compared with CK3 group (3.42±0.10kg / cm), 2 The level was significantly increased by 12.6% (P<0.01) compared to the CK2 group (3.61±0.11 kg / cm²). 2The average eggshell thickness was 0.38±0.02 mm, significantly higher than that of the CK3 group (0.34±0.02 mm) by 11.8% (P<0.05). Scanning electron microscopy showed that the papillary layer of the eggshell in the experimental group was dense and uniform, the interpapillary spacing was reduced by 30%, and the palisade layer thickness increased by 15%. The calcium content of the eggshell was 38.5±1.2%, and the phosphorus content was 2.1±0.1%, which were 5.2% and 9.5% higher than those in the CK3 group, respectively (P<0.05). Vitamin D2 converted from ergosterol activated the expression of calcium-binding protein (CaBP-D28k) in the laying hen's intestine, promoting calcium and phosphorus deposition in the eggshell. At the same time, the short-chain fatty acids produced by the probiotic metabolism in the fermentation products improved the intestinal pH, enhanced the calcium and phosphorus absorption efficiency, and synergistically improved the density and mechanical strength of the eggshell.

[0145] (3) Nutritional fortification effect of egg yolk

[0146] Comparison table of nutritional fortification effects of egg yolks

[0147]

[0148] The 25-hydroxyvitamin D3 content in the egg yolk of the experimental group reached 18.6±1.5 ng / g, which was significantly increased by 353.7% compared with the CK3 group (4.1±0.5 ng / g) (P<0.01) and significantly increased by 45.3% compared with the CK2 group (12.8±1.2 ng / g) (P<0.05); the α-tocopherol content reached 28.6±2.1 μg / g, which was significantly increased by 55.4% compared with the CK3 group (18.4±1.8 μg / g) (P<0.01), significantly increased by 28.3% compared with the CK2 group (22.3±1.9 μg / g) (P<0.05), and significantly increased by 76.5% compared with the CK1 group (16.2±1.5 μg / g) (P<0.01). It is evident that the solid-state fermentation process of this invention avoids the oxidative loss of vitamin E in traditional chemical methods (retention rate >92%). At the same time, the fermentation process of *Penicillium cicadae* protects the ester bonds of vitamin E, thereby improving its deposition efficiency in laying hens. Ergosterol is metabolized by laying hens into vitamin D2, and then further converted into the active form 25-hydroxyvitamin D3, significantly enhancing the nutritional value of egg yolks.

[0149] This invention utilizes key technologies such as the resource utilization of deodorized tea seed oil distillate, two-step sequential fermentation of yeast and *Penicillium cicadae*, cascade regulation of the endogenous MVA pathway, and precise compounding of nutrients for laying hens to form a functional feed additive preparation system with independent intellectual property rights. The technical solution includes raw material pretreatment, fermentation process, regulation strategies, post-processing methods, compound formulation, and application in laying hen farming. Any equivalent substitutions or reasonable parameter adjustments based on the core ideas of this invention should be included within the scope of protection.

Claims

1. A method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate, characterized in that, Includes the following steps: (1) Raw material pretreatment: The deodorized distillate of tea seed oil is heated to 60-65℃ and centrifuged at a speed of 6000-8000 rpm to remove impurities and obtain a clear oily liquid. (2) First step: Yeast liquid fermentation: Using the pretreated deodorized distillate as the main carbon source, a fermentation medium was prepared, and Candida utilis or Saccharomyces cerevisiae seed liquid was inoculated. Fermentation was carried out at 30-37℃, with an aeration rate of 0.5-0.8 volumes / (volume·min) and a stirring speed of 300-500 rpm for 36-48 hours until the free fatty acid content was ≤8 g / L and the glycerol ester hydrolysis rate was ≥75%. (3) Second step: Solid-state fermentation of *Penicillium cicadae*: The fermentation product from the first step is mixed with wheat bran, rice husks, soybean meal, etc., to prepare a solid substrate. A suspension of *Penicillium cicadae* spores is then inoculated into the substrate. Fermentation is carried out for 72-96 hours through a three-stage cascade regulation method: mycelial proliferation period - metabolic flow regulation period - product stabilization period. The cascade regulation measures include: nitrogen restriction treatment after 24 hours of fermentation, controlling the iron citrate content in the substrate to 0.01%-0.03% to achieve iron restriction, performing dissolved oxygen pulse operation every 12 hours, lowering the temperature to 24-26℃ after 48 hours of fermentation, and adjusting the pH value to 5.0-5.5 after 36 and 60 hours of fermentation. (4) Fermentation product processing: The solid fermentation product is placed at 45-50℃ for vacuum drying until the moisture content is ≤8%. Then it is subjected to ultra-fine pulverization and passed through a 60-80 mesh sieve to obtain the fermentation core material. (5) Compound preparation: According to the weight percentage, 40%-50% of fermentation core material, 25%-30% of stone powder, 10%-15% of dicalcium phosphate, 8%-12% of zeolite powder, 2%-4% of yeast cell wall polysaccharide and 0.02%-0.05% of ethoxyquinoline are uniformly mixed to obtain the laying hen feed additive.

2. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: The *Candida utilis* strain used in step (2) is *Candida utilis* AS2.1180, and the *Saccharomyces cerevisiae* strain is *Saccharomyces cerevisiae* CICC 1001. The fermentation medium formula is as follows: 100-150 g / L deodorized distillate, 5-8 g / L (NH4)2SO4, 0.5-1.0 g / L MgSO4·7H2O, 2-3 g / L KH2PO4, and an initial pH of 5.5-6.

0. The inoculum size of the seed culture is 8-12% (v / v), and the concentration of the seed culture cells is not less than 1×10⁻⁶. 8 CFU / mL.

3. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: The Paecilomycescicadae in step (3) is CGMCC 3.3790; the solid substrate formula (by dry weight percentage) is: 45-55% yeast fermentation broth, 20-30% wheat bran, 10-15% rice husk, 8-12% soybean meal, 0.01-0.03% ferric citrate, 0.5-1.0% KH2PO4, 0.1-0.2% MgSO4·7H2O, and the substrate moisture content is 58-62%.

4. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: The concentration of the spore suspension in step (3) is 1×10⁻⁶. 8 -5×10 8 The inoculum concentration is 5-8% (v / w), and a solution containing 0.05% Tween-80 is sprayed during inoculation. The temperature during the mycelial proliferation phase is 25-28℃, and the temperature during the product stabilization phase is 26-28℃.

5. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: In step (2), the extracellular lipase activity during yeast fermentation is not less than 50 U / mL, and sterol esters are converted into free phytosterols through enzymatic hydrolysis.

6. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: The cascade regulation in step (3) does not require the addition of exogenous mevalonic acid. By regulating the process parameters, the activity of HMG-CoA reductase is increased by 2.3-3.1 times, and the transcriptional level of mevalonic acid kinase is upregulated by 4.5 times.

7. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: At the end of fermentation in step (3), the ergosterol content shall not be less than 0.50 mg / g (dry basis), the total sterol retention rate shall not be less than 90%, the mycelial biomass shall not be less than 0.40 g / g dry basis, and the free fatty acid residue shall not be higher than 2.5 g / kg.

8. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: The compounded product in step (5) meets the following indicators: vitamin D not less than 1600 IU / kg, vitamin E not less than 5 IU / kg, crude protein not less than 16.5%, calcium content 3.5%, phosphorus content 0.6%, and probiotic live bacteria count not less than 2×10⁻⁶. 9 CFU / g.

9. The method for preparing laying hen feed additives by fermentation of deodorized tea seed oil distillate according to claim 1, characterized in that: In step (4), the fermentation product is directly dried and pulverized without separation and purification. After the mycelium of *Penicillium cicadae* breaks naturally, the intracellular ergosterol release rate is greater than 85%.

10. The application of the layer hen feed additive prepared according to any one of claims 1-9 in layer hen farming, characterized in that: The additive is added to the daily diet of laying hens at a rate of 0.2-0.5%, which can improve egg production rate, improve eggshell quality, increase the vitamin D3 content of egg yolks, and regulate intestinal flora.

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