Method for improving oxidation resistance of maggot and synthesizing GSH

Abstract

The invention relates to the technical field of medicinal breeding of special insects, in particular to a method for improving oxidation resistance of maggot and synthesizing GSH (glutathione), which comprises the following steps: preparing pig lung, medlar and salvia miltiorrhiza fermented feed; on the basis of the fermented feed, continuous three-generation directional stress screening is carried out, offspring is used as a next-generation provenance, and a target maggot population is obtained. The fermented feed containing active ingredients such as lycium barbarum polysaccharide and tanshinone IIA is prepared, continuous three-generation directional screening is matched, a GSH synthesis pathway in maggot bodies is activated, expression of antioxidant related substances is enhanced, the oxidative damage resistance of maggots is remarkably improved, meanwhile, populations are endowed with prominent functional application value, and the application prospect is wide. The method can be widely applied to research and development of antioxidant skin care products and other products, and can also provide natural biological resource support for auxiliary improvement of oxidative stress related diseases.

Description

Technical Field

[0001] This invention relates to the field of medicinal insect breeding technology, specifically a method for enhancing the antioxidant capacity and GSH synthesis of the grain insect. Background Technology

[0002] In recent years, the field of special insect aquaculture has gradually focused on combining feed composition optimization with functional component enhancement. Pig lungs, rich in cysteine ​​(a GSH precursor) and ω-3 fatty acids, have attracted attention as a potential feed ingredient for insect farming. Meanwhile, the antioxidant value of active ingredients from traditional Chinese medicines such as wolfberry polysaccharides, β-carotene, and tanshinone IIA from danshen has been widely recognized. The former can activate the Nrf2 antioxidant pathway, while the latter promotes the expression of γ-glutamylcysteine ​​synthase genes. These findings provide important references for improving the antioxidant function of insects through feed regulation. As a special insect with both nutritional value and functional development potential, the activity of glutamylcysteine ​​synthase and the content of antioxidant peptides such as GPx homologs in the *Gnaphalium affine* have become core indicators of industry focus.

[0003] In current technologies related to the breeding of special insects, the *Pteris vittata* (a type of insect) faces significant bottlenecks in functional enhancement. Its γ-glutamylcysteine ​​synthase activity is generally low, typically not exceeding 0.5 U / mg, directly limiting GSH synthesis efficiency. Simultaneously, insufficient expression of antioxidant peptides hinders effective enhancement of antioxidant function. Although the nutritional advantages of pig lungs and the active ingredient characteristics of wolfberry and salvia miltiorrhiza have been confirmed, existing technologies have failed to organically link the nutritional supply of pig lungs with the functional components of traditional Chinese medicine. Furthermore, feed fermentation processes have not yet been implemented to synergize the nutritional and functional properties of different raw materials, thereby specifically addressing the key issues of insufficient GSH synthesis and antioxidant expression in *Pteris vittata*. Summary of the Invention

[0004] The purpose of this invention is to provide a pig lung-goji-tanshinone co-fermented feed, which activates the Nrf2 pathway through goji polysaccharide, promotes γ-GCS expression through tanshinone IIA, and enhances the GSH content and antioxidant peptide GPx level of the five-grain insect through three generations of domestication.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] A method for enhancing the antioxidant capacity and GSH synthesis of *Pteris vittata* (a type of insect), comprising the following steps:

[0007] (1) Preparation of pig lung-wort-salvia miltiorrhiza fermented feed;

[0008] (2) Based on fermented feed, three consecutive generations of targeted stress screening were conducted, and the offspring were used as the breeding source for the next generation to obtain the target grain insect population; the three consecutive generations of targeted stress screening were conducted in the following manner:

[0009] First-generation screening: Larvae were fed fermented feed and subjected to oxidative stress to screen for individuals with glutamylcysteine ​​synthase activity ≥0.8 U / mg;

[0010] Second-generation screening: Larvae were fed fermented feed supplemented with tanshinone IIA and subjected to oxidative stress by applying a higher concentration of hydrogen peroxide. Individuals with glutathione peroxidase activity ≥120 U / mg were screened.

[0011] Third-generation screening: Larvae were fed fermented feed and subjected to high sugar stress. Individuals with glutathione ≥900 μg / g and glutathione peroxidase ≥150 U / mg were selected.

[0012] Furthermore, in step (1), the preparation of pig lung-wort-salvia miltiorrhiza fermented feed specifically includes the following steps:

[0013] S1. Raw material pretreatment: Take 80% fresh pig lung, 12% dried wolfberry, 5% dried salvia miltiorrhiza, 3% wheat bran and 0.5% ascorbic acid by weight percentage;

[0014] S11. Pig lung processing: Fresh pig lung is chopped and homogenized. After homogenization, the particle size is ≤1mm to obtain pig lung homogenate.

[0015] S12. Preparation of wolfberry supernatant: The dried wolfberries are crushed and passed through a 40-mesh sieve. They are mixed with water at a ratio of 1:10 and decocted at 60°C for 3 hours, or mixed with water at a ratio of 1:5 and extracted at 85°C for 2 hours. The supernatant is then filtered to obtain wolfberry supernatant.

[0016] S13. Preparation of Danshen-Wheat Bran Mixture: After pulverizing dried Danshen, it is premixed with wheat bran in a 1:1 ratio to obtain Danshen-Wheat Bran Mixture;

[0017] S2, Mixed Fermentation:

[0018] S21. Material mixing: First, place the pig lung homogenate in a mixing tank, slowly add the wolfberry supernatant and stir, then add the salvia miltiorrhiza-wheat bran mixture and continue stirring until uniform;

[0019] S22. Add antioxidant: Add ascorbic acid to the mixture obtained in S21;

[0020] S23. Inoculation strain: Add fermentation strain to the material obtained in S22. The fermentation strain is a combination of 3% yeast and 1% acetic acid bacteria, or a combination of 3% lactic acid bacteria and 2% Bacillus subtilis.

[0021] S24. Fermentation control: When the fermentation strain is a combination of yeast and acetic acid bacteria, it is placed in an environment of 25℃ for aerobic fermentation for 36 hours, and the final pH of fermentation is ≤5.5; when the fermentation strain is a combination of lactic acid bacteria and Bacillus subtilis, it is sealed in a tank and placed in an environment of 28℃ for anaerobic fermentation, and the final pH of fermentation is ≤4.7 and stabilized for more than 12 hours.

[0022] The fermented product obtained contained ≥1.8% Lycium barbarum polysaccharide, ≥0.3% Tanshinone IIA, and ≥1.8g / kg cysteine.

[0023] Further, in step S24, the monitoring process for mixed fermentation is as follows: samples are taken every 6 hours during fermentation, the pH of the mixture is measured using a calibrated pH meter, the measured values ​​are recorded, and a pH change curve is plotted; when the fermentation strain is a combination of yeast and acetic acid bacteria, the monitoring continues until the pH ≤ 5.5; when the fermentation strain is a combination of lactic acid bacteria and Bacillus subtilis, the monitoring continues until the pH ≤ 4.7 and remains stable for more than 12 hours.

[0024] Furthermore, the seed preparation process for each generation is as follows: adult insects are induced to lay eggs in a concentrated manner for 1-2 hours using inducing feed. The inducing feed consists of 80% fermented feed, 5% yeast extract, 5% glucose, 5% wolfberry polysaccharide solution, and 5% vitamin mixture by weight percentage. After mixing, the moisture content is adjusted to 60%. A dark environment is maintained during the induction of egg laying. The eggs are then disinfected before hatching.

[0025] Furthermore, oxidative stress is achieved by adding hydrogen peroxide to the feed. In the first-generation screening, the final concentration of hydrogen peroxide is 0.01%, applied once daily. In the second-generation screening, the final concentration of hydrogen peroxide is 0.03%, applied continuously on days 2-3 of the second-year age. The high-sugar stress is achieved by adding sucrose to the feed, with a sucrose content of 8%, applied continuously on days 3-5 of the third-year age.

[0026] Furthermore, the amount of tanshinone IIA added to the feed used in the second-generation screening was 0.05%.

[0027] Furthermore, the glutathione content detection process is as follows: after homogenizing the sample, the protein is precipitated with 5% sulfosalicylic acid, and the absorbance at 412 nm is measured using the DTNB method to calculate the glutathione content per μmol / g of protein.

[0028] Furthermore, the detection process for glutathione peroxidase activity is as follows: using the NADPH consumption rate method, the amount of NADPH consumed per minute is measured using a kit, and the glutathione peroxidase activity per U / mg protein is calculated.

[0029] Furthermore, the detection process for the expression of relevant antioxidant genes is as follows: RNA is extracted from the sample, cDNA is synthesized using the extracted RNA as a template, and qPCR is used for detection. The primers used for qPCR are targeted at the GPx gene and the GCLC gene, with β-actin as an internal control.

[0030] The advantages of this invention compared to the prior art are:

[0031] 1. This invention enriches wolfberry polysaccharides in customized fermented feed, and activates the Nrf2 pathway in the body of the grain insect in a targeted manner in conjunction with other nutrients in the raw materials. Combined with artificial selection pressure through three generations of stress screening, it achieves a significant increase of γ-GCS activity by 60%, thereby promoting a significant increase in GSH synthesis efficiency. Ultimately, the GSH content in the grain insect is twice that of conventionally farmed individuals.

[0032] 2. This invention achieves a significant increase in the repair rate of oxidative damage caused by H2O2 from 50% in wild species to 85% through precise supply of tanshinone IIA in fermented feed, thereby promoting the expression and activity of the GPx gene in the insect. After screening and enriching individuals with strong stress resistance through gradient oxidative stress, the overall antioxidant defense capability is significantly improved, resulting in a qualitative leap in the overall antioxidant defense capability and better resistance to external oxidative stress.

[0033] 3. This invention obtains a population of grain insects with high GSH and high GPx activity through targeted cultivation. The natural antioxidant components enriched in the insects have stable activity and achieve outstanding functional application value. The high content of GSH can effectively scavenge free radicals, making it suitable for the research and development and production of antioxidant skin care products. The high activity of GPx can alleviate oxidative stress-related diseases and provide high-quality natural biological resource support for the auxiliary improvement of liver damage and other diseases. Attached Figure Description

[0034] The accompanying drawings, which form part of this application, are used to provide a further understanding of the application and to make other features, objects, and advantages of the application more apparent. The illustrative embodiments and descriptions of this application are used to explain the application and do not constitute an undue limitation of the application.

[0035] In the attached diagram:

[0036] Figure 1 This data serves as verification of the trait stability of the final population after three generations of continuous reproduction in Example 1. Detailed Implementation

[0037] The detailed description of the following embodiments is used to exemplify the principles of this application, but should not be used to limit the scope of this application. That is, the method of improving the antioxidant capacity and GSH synthesis of the five grain insects in this application is not limited to the described embodiments.

[0038] The present invention will be further described below with reference to embodiments.

[0039] Example 1

[0040] A method for enhancing the antioxidant capacity and GSH synthesis of *Pteris vittata* (a type of insect), comprising the following steps:

[0041] (1) Preparation of pig lung-wort-salvia miltiorrhiza fermented feed;

[0042] (2) Based on fermented feed, three consecutive generations of targeted stress screening were conducted, and the offspring were used as the breeding source for the next generation to obtain the target grain insect population; the three consecutive generations of targeted stress screening were conducted in the following manner:

[0043] First-generation screening: Larvae were fed fermented feed and subjected to oxidative stress to screen for individuals with glutamylcysteine ​​synthase activity ≥0.8 U / mg;

[0044] Second-generation screening: Larvae were fed fermented feed supplemented with tanshinone IIA and subjected to oxidative stress by applying a higher concentration of hydrogen peroxide. Individuals with glutathione peroxidase activity ≥120 U / mg were screened.

[0045] Third-generation screening: Larvae were fed fermented feed and subjected to high sugar stress. Individuals with glutathione ≥900 μg / g and glutathione peroxidase ≥150 U / mg were selected.

[0046] In this specific embodiment, the core of the entire method lies in the precise coordination of feed formulation and targeted screening. A customized fermented feed provides the nutritional foundation, and then gradient stress screening enriches desirable traits. An induction feed containing 80% fermented feed, 5% yeast extract, 5% glucose, 5% wolfberry polysaccharide solution, and 5% vitamin mixture is selected. After mixing, the moisture content is adjusted to 60%. This ratio maximizes the attraction of adult insects to concentrate egg-laying. The 1-2 hour induction period ensures sufficient egg production while preventing excessive egg accumulation. The dark environment matches the egg-laying habits of the grain beetle, further increasing egg concentration. The collected eggs are disinfected before hatching. Simultaneous hatching of larvae ensures consistent screening conditions and reduces errors caused by individual differences.

[0047] In the first generation of selection, larvae were fed a customized fermented feed throughout the entire process. The application of oxidative stress established initial pressure for trait selection. After nutritional supply and stress induction during the rearing period, individuals with glutamylcysteine ​​synthase activity meeting the standard were precisely selected. 2500 individuals meeting the standard of ≥0.8 U / mg had their key enzyme system for GSH synthesis preliminarily activated, and the superior traits of the population were gradually strengthened. The second generation of selection continued to use the superior breeding stock of the first generation. The feed supplemented with tanshinone IIA could specifically enhance enzyme gene expression. Higher concentrations of hydrogen peroxide stress further selected individuals with stronger stress resistance. The glutathione peroxidase activity standard of ≥120 U / mg ensured that the selected individuals had a more complete antioxidant defense system. At this time, the enzyme activity was 100% higher than that of the wild species, and the antioxidant capacity of the population achieved a significant leap. The third-generation screening used high-sugar stress to simulate nutritional stress in the natural environment, forming a gradient progression with the first two generations of screening. The final 1,000 target individuals selected achieved dual compliance with the core antioxidant indicators: glutathione ≥ 950 μg / g and glutathione peroxidase ≥ 160 U / mg.

[0048] Furthermore, in step (1), the preparation of pig lung-wort-salvia miltiorrhiza fermented feed specifically includes the following steps:

[0049] S1. Raw material pretreatment: Take 80% fresh pig lung, 12% dried wolfberry, 5% dried salvia miltiorrhiza, 3% wheat bran and 0.5% ascorbic acid by weight percentage;

[0050] S11. Pig lung processing: Fresh pig lung is chopped and homogenized. After homogenization, the particle size is ≤1mm to obtain pig lung homogenate.

[0051] S12. Preparation of wolfberry supernatant: The dried wolfberries are crushed and passed through a 40-mesh sieve. They are mixed with water at a ratio of 1:10 and decocted at 60°C for 3 hours, or mixed with water at a ratio of 1:5 and extracted at 85°C for 2 hours. The supernatant is then filtered to obtain wolfberry supernatant.

[0052] S13. Preparation of Danshen-Wheat Bran Mixture: After pulverizing dried Danshen, it is premixed with wheat bran in a 1:1 ratio to obtain Danshen-Wheat Bran Mixture;

[0053] S2, Mixed Fermentation:

[0054] S21. Material mixing: First, place the pig lung homogenate in a mixing tank, slowly add the wolfberry supernatant and stir, then add the salvia miltiorrhiza-wheat bran mixture and continue stirring until uniform;

[0055] S22. Add antioxidant: Add ascorbic acid to the mixture obtained in S21;

[0056] S23. Inoculation strain: Add fermentation strain to the material obtained in S22. The fermentation strain is a combination of 3% yeast and 1% acetic acid bacteria, or a combination of 3% lactic acid bacteria and 2% Bacillus subtilis.

[0057] S24. Fermentation control: When the fermentation strain is a combination of yeast and acetic acid bacteria, it is placed in an environment of 25℃ for aerobic fermentation for 36 hours, and the final pH of fermentation is ≤5.5; when the fermentation strain is a combination of lactic acid bacteria and Bacillus subtilis, it is sealed in a tank and placed in an environment of 28℃ for anaerobic fermentation, and the final pH of fermentation is ≤4.7 and stabilized for more than 12 hours.

[0058] The fermented product obtained contained ≥1.8% Lycium barbarum polysaccharide, ≥0.3% Tanshinone IIA, and ≥1.8g / kg cysteine.

[0059] In a specific embodiment, the raw material ratio was designed with precise consideration. 80% fresh pig lungs can provide sufficient cysteine, which is the core precursor of GSH synthesis. 12% dried wolfberry and 5% dried tanshinone bring functional components such as wolfberry polysaccharides and tanshinone IIA. 3% wheat bran can optimize the physical structure of the feed, and 0.5% ascorbic acid can effectively inhibit lipid oxidation during fermentation and protect the active ingredients from being destroyed.

[0060] In the raw material pretreatment stage, fresh pig lungs are chopped and homogenized to a particle size ≤1mm. This treatment increases the contact area between the raw material and the bacteria and enzymes, improving the release efficiency of nutrients and making it easier for the larvae of the grainworm to digest and absorb. Dried wolfberries are pulverized through a 40-mesh sieve to ensure that the active ingredients are fully dissolved during the extraction process. The mixture is mixed with water at a ratio of 1:5 and extracted at 85℃ for 2 hours. This ensures the extraction rate of wolfberry polysaccharides while avoiding degradation of components due to prolonged high-temperature processing. The supernatant after filtration concentrates the core functional components of wolfberries. Dried salvia miltiorrhiza is pulverized and premixed with wheat bran at a 1:1 ratio. The addition of wheat bran prevents the salvia miltiorrhiza powder from clumping, ensuring its even distribution in the mixed feed and allowing each larva to ingest a balanced amount of functional components.

[0061] During the mixed fermentation stage, the sequential mixing of raw materials avoids excessively high local component concentrations, which could affect the fermentation effect. The timing of ascorbic acid addition ensures that it exerts its antioxidant effect in the early stages of fermentation. The combination of 3% lactic acid bacteria and 2% Bacillus subtilis works together to decompose macromolecules in the raw materials, converting them into small peptides and amino acids. The anaerobic fermentation environment at 28℃ matches the growth habits of the microorganisms, maximizing fermentation efficiency. Fermentation to pH ≤ 4.7 and stabilization for more than 12 hours ensures the stability of the fermentation product and avoids incomplete or over-fermentation affecting feed quality. Final testing showed that the content of wolfberry polysaccharides was 1.9%, tanshinone IIA was 0.35%, and cysteine ​​was ≥ 1.8 g / kg, all of which were better than the preset standards. Such fermentation products can provide sufficient nutritional precursors and functional components for the grain insects, directly supporting the improvement of antioxidant capacity.

[0062] Further, in step S24, the monitoring process for mixed fermentation is as follows: samples are taken every 6 hours during fermentation, the pH of the mixture is measured using a calibrated pH meter, the measured values ​​are recorded, and a pH change curve is plotted; when the fermentation strain is a combination of yeast and acetic acid bacteria, the monitoring continues until the pH ≤ 5.5; when the fermentation strain is a combination of lactic acid bacteria and Bacillus subtilis, the monitoring continues until the pH ≤ 4.7 and remains stable for more than 12 hours.

[0063] In this specific embodiment, pH monitoring during fermentation is a crucial step in ensuring feed quality. Sampling every 6 hours accurately captures pH trends, preventing fermentation from spiraling out of control due to untimely monitoring. Using a calibrated pH meter ensures the accuracy of the data, while plotting pH curves visually reflects the fermentation process, facilitating the assessment of the microbial metabolic state. Continuous monitoring until the pH drops to 4.7 and remains stable for more than 12 hours ensures complete fermentation. At this point, harmful microorganisms in the fermentation products are effectively inhibited, and the content of functional components reaches its peak, providing a stable and reliable feed supply for livestock farming.

[0064] Furthermore, the seed preparation process for each generation is as follows: adult insects are induced to lay eggs in a concentrated manner for 1-2 hours using inducing feed. The inducing feed consists of 80% fermented feed, 5% yeast extract, 5% glucose, 5% wolfberry polysaccharide solution, and 5% vitamin mixture by weight percentage. After mixing, the moisture content is adjusted to 60%. A dark environment is maintained during the induction of egg laying. The eggs are then disinfected before hatching.

[0065] In this specific embodiment, a standardized process for preparing the breeding stock is a prerequisite for ensuring screening effectiveness. The formulation of the induction feed balances nutritional appeal and functionality. 80% fermented feed allows adult insects to adapt to the larval feed composition in advance, while 5% yeast extract and glucose provide a rapid energy source. The mixture of wolfberry polysaccharide solution and vitamins enhances the physical condition of adult insects, thereby improving egg quality. The moisture content is adjusted to 60% after mixing, which meets the humidity requirements for adult egg laying while preventing excessive moisture that could lead to spoilage. The 1-2 hour induction period has been optimized through multiple trials to obtain a sufficient number of eggs in the shortest possible time. The dark environment reduces external interference, allowing adult insects to concentrate on egg laying. After disinfection, the eggs hatch, effectively reducing the risk of pests and diseases, ensuring the health and purity of the breeding stock, and providing a high-quality basic population for targeted screening.

[0066] Furthermore, oxidative stress is achieved by adding hydrogen peroxide to the feed. In the first-generation screening, the final concentration of hydrogen peroxide is 0.01%, applied once daily. In the second-generation screening, the final concentration of hydrogen peroxide is 0.03%, applied continuously on days 2-3 of the second-year age. The high-sugar stress is achieved by adding sucrose to the feed, with a sucrose content of 8%, applied continuously on days 3-5 of the third-year age.

[0067] In specific embodiments, the design of stress conditions follows a gradient progression principle to ensure that population traits are gradually strengthened rather than abruptly changed. In the first-generation screening, a final hydrogen peroxide concentration of 0.01% and an application frequency of once daily activate the larval antioxidant enzyme system without causing mass mortality due to excessive concentration, thus preserving a sufficient number of superior individuals for the population. In the second-generation screening, the final hydrogen peroxide concentration is increased to 0.03% and continuously applied on days 2-3 of the second instar. At this time, the larvae are in a critical stage of growth and development and are more sensitive to stress responses. Continuous stress further selects individuals with stronger stress resistance, making the population traits more stable. High sugar stress is applied on days 3-5 of the third instar, with an 8% sucrose content simulating high sugar stress in the natural environment. Larvae at this stage have vigorous metabolism, can quickly respond to stress and adjust their own metabolic mechanisms, inducing a coordinated response between sugar metabolism and the antioxidant system, ultimately achieving a stable increase in core antioxidant indicators.

[0068] Furthermore, the amount of tanshinone IIA added to the feed used in the second-generation screening was 0.05%.

[0069] In this specific embodiment, the addition of 0.05% tanshinone IIA was precisely calculated to effectively promote γ-GCS gene expression and enhance the GSH synthesis pathway without causing toxicity to larvae due to excessive concentration. Thoroughly mixing tanshinone IIA with the basal fermented feed ensures that each larva ingests a balanced amount of functional components, avoiding deviations in screening results due to localized concentration differences. This method of addition maximizes the effect of tanshinone IIA, synergizing with higher concentrations of oxidative stress to further enhance the population's antioxidant capacity and help achieve dual-indicator targets in the third-generation screening.

[0070] Furthermore, the glutathione content detection process is as follows: after homogenizing the sample, the protein is precipitated with 5% sulfosalicylic acid, and the absorbance at 412 nm is measured using the DTNB method to calculate the glutathione content per μmol / g of protein.

[0071] In a specific embodiment, the detection process is designed to accurately capture the true glutathione content. Sample homogenization breaks down cell structure, allowing for the full release of internal GSH. The use of 5% sulfosalicylic acid effectively precipitates proteins, preventing interference with the detection results. The DTNB method boasts high specificity and sensitivity; the 412nm detection wavelength accurately captures the absorbance of the reaction products. The glutathione content calculated using a standard curve objectively reflects the GSH synthesis level of the target population. The detection results show that the glutathione content of the target population is 950 μg / g, a 217% increase compared to the wild-type population's 300 μg / g, directly demonstrating the combined effect of feed formulation and targeted screening.

[0072] Furthermore, the detection process for glutathione peroxidase activity is as follows: using the NADPH consumption rate method, the amount of NADPH consumed per minute is measured using a kit, and the glutathione peroxidase activity per U / mg protein is calculated.

[0073] In a specific embodiment, the NADPH consumption rate method can reflect the catalytic activity of GPx in real time. The use of a dedicated kit ensures the accuracy and repeatability of the detection. The amount of NADPH consumed per minute is directly related to enzyme activity, and the converted U / mg protein units facilitate cross-sectional comparisons between different populations. The detection results show that the GPx activity of the target population is 160 U / mg, which is 100% higher than the 80 U / mg of the wild species. This indicates that after three generations of targeted screening, the antioxidant enzyme activity of the population has been significantly enhanced, and the antioxidant defense system has become more complete.

[0074] Furthermore, the detection process for the expression of relevant antioxidant genes is as follows: RNA is extracted from the sample, cDNA is synthesized using the extracted RNA as a template, and qPCR is used for detection. The primers used for qPCR are targeted at the GPx gene and the GCLC gene, with β-actin as an internal control.

[0075] In this specific embodiment, gene-level detection can fundamentally verify the mechanism of trait enhancement. The RNA extraction process ensures the integrity and purity of nucleic acids, providing a high-quality template for reverse transcription. Using RNA as a template to synthesize cDNA solves the problem of RNA degradation, providing a stable amplification template for qPCR detection. The specific primers for qPCR can accurately target the GPx and GCLC genes, while the use of β-actin as an internal control corrects for errors in sample processing, ensuring the accuracy of gene expression fold increases. The detected GPx gene expression fold increase was 2.8 ± 0.3, confirming the high expression state of this gene at the molecular level and explaining the intrinsic reason for the enhanced antioxidant capacity of the target population.

[0076] like Figure 1 As shown, the target grain insect population obtained through the above method, after continuous breeding to the sixth generation, exhibited a coefficient of variation of less than 5% for core indicators, indicating stable trait inheritance. Verification of trait stability is crucial for large-scale application of the population. Using the F3 generation as the basic parent, continuous breeding was conducted to the F6 generation, maintaining the same rearing and selection conditions for each generation to ensure the objectivity of the verification results. Data from the F3 to F6 generations showed that the fluctuation range of GSH content, GPx activity, and GPx gene expression fold change was extremely small, with coefficients of variation all less than 5%. This stability indicates that the superior traits obtained through three generations of targeted selection have been stably inherited, and the population will not experience trait degeneration with increasing generations, providing a reliable guarantee for large-scale breeding and industrial application.

[0077] It should be noted that the combination of the technical features in this case is not limited to the combination methods described in the claims of this case or the combination methods described in the specific embodiments. All technical features described in this case can be freely combined or combined in any way, unless they contradict each other.

[0078] It should also be noted that the embodiments listed above are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and similar changes or modifications made thereto are those that can be directly derived or easily conceived by those skilled in the art from the content disclosed in the present invention, and should all fall within the protection scope of the present invention.

Claims

1. A method for improving the antioxidant capacity and GSH synthesis of silkworms, characterized in that, Comprise the following steps: Preparation of pig lung- medlar- salvia fermentation feed; With the fermentation feed as the basis, continuous three generations of directional stress screening is carried out, and the offspring is used as the seed source of the next generation to obtain the target five grainworm populations; The continuous three generations of directional stress screening is carried out in the following manner: First generation screening: the larvae are fed with the fermentation feed, oxidative stress is applied, and individuals with glutamyl cysteine synthetase activity ≥0.8 U / mg are screened; Second generation screening: the larvae are fed with the fermentation feed added with tanshinone II A, higher concentration of hydrogen peroxide is applied for oxidative stress, and individuals with glutathione peroxidase activity ≥120 U / mg are screened; Third generation screening: the larvae are fed with the fermentation feed, high sugar stress is applied, and individuals with glutathione ≥900 μg / g and glutathione peroxidase ≥150 U / mg are screened.

2. The method of claim 1, wherein the method is characterized by: In step (1), the preparation of the pig lung- medlar- salvia fermentation feed specifically comprises the following steps: S1, raw material pretreatment: take 80% of fresh pig lung, 12% of dried medlar, 5% of dried salvia, 3% of wheat bran and 0.5% of ascorbic acid by mass percentage; S11, pig lung treatment: cut the fresh pig lung into pieces and homogenize, the particle size after homogenization is ≤1mm, and pig lung homogenate is obtained; S12, preparation of medlar supernatant: crush the dried medlar to pass through a 40 mesh sieve, mix with water at a solid-liquid ratio of 1:10 and cook at 60℃ for 3 hours, or mix with water at a solid-liquid ratio of 1:5 and extract at 85℃ for 2 hours, filter and take the supernatant to obtain the medlar supernatant; S13, preparation of salvia- wheat bran mixture: crush the dried salvia and uniformly premix with wheat bran at a ratio of 1:1 to obtain a salvia- wheat bran mixture; S2, mixed fermentation: S21, material mixing: first place the pig lung homogenate in a stirring tank, slowly add the medlar supernatant and stir, then add the salvia- wheat bran mixture and continue to stir until uniform; S22, addition of antioxidant: add ascorbic acid to the mixture obtained in S21; S23, inoculation of bacteria: add fermentation bacteria to the material obtained in S22, the fermentation bacteria are a combination of 3% of yeast and 1% of acetic acid bacteria, or a combination of 3% of lactic acid bacteria and 2% of bacillus subtilis; S24, fermentation control: when the fermentation bacteria are a combination of yeast and acetic acid bacteria, place in an aerobic fermentation environment at 25℃ for 36 hours, and the final pH is ≤5.5; when the fermentation bacteria are a combination of lactic acid bacteria and bacillus subtilis, place in an anaerobic fermentation environment at 28℃ after sealing the tank, and the final pH is ≤4.7 and stable for more than 12 hours; The fermentation product prepared has a medlar polysaccharide content ≥1.8%, a tanshinone II A content ≥0.3%, and a cysteine content ≥1.8g / kg.

3. The method of claim 2, wherein the method is characterized by: In step S24, the monitoring process of the mixed fermentation is as follows: sampling every 6 hours during the fermentation process, measuring the pH of the mixture by using the calibrated pH meter, recording the measured value and drawing the pH change curve; when the fermentation strain is a combination of yeast and acetic acid bacteria, until pH≤5.5; when the fermentation strain is a combination of lactic acid bacteria and Bacillus subtilis, until pH≤4.7 and stable for more than 12 hours.

4. The method of claim 3, wherein the method is characterized by: The preparation process of each generation of seed source is as follows: using induced feed to induce adult insects to concentrate oviposition for 1-2 hours, the induced feed is 80% fermented feed, 5% yeast extract, 5% glucose, 5% wolfberry polysaccharide solution and 5% vitamin mixed solution by mass percentage, after mixing, adjust the moisture content to 60%, maintain a dark environment during the oviposition induction period; the eggs are sterilized and hatched.

5. The method of claim 4, wherein the method is characterized by: The oxidative stress is adding hydrogen peroxide in the feed, the final concentration of hydrogen peroxide in the first generation screening is 0.01%, which is applied once a day; the final concentration of hydrogen peroxide in the second generation screening is 0.03%, which is continuously applied from the 2nd to 3rd day of the 2nd generation; the high sugar stress is adding sucrose in the feed, the sucrose content is 8%, which is continuously applied from the 3rd to 5th day of the 3rd generation.

6. The method of claim 5, wherein the method is characterized by: The addition amount of tanshinone ⅡA in the feed used in the second generation screening is 0.05%.

7. The method of claim 6, wherein the antioxidant capacity and GSH synthesis of the larvae are improved. The detection process of glutathione content is as follows: after homogenizing the sample, precipitate the protein with 5% sulfosalicylic acid, measure the absorbance at 412 nm by using DTNB method, and calculate the glutathione content of μmol / g protein.

8. The method of claim 7, wherein the antioxidant capacity and GSH synthesis of the larvae are improved. The detection process of glutathione peroxidase activity is as follows: using NADPH consumption rate method, using kit to measure the amount of NADPH consumed per minute, and converting to get the glutathione peroxidase activity of U / mg protein.

9. The method of claim 8, wherein the method is characterized by: The detection process of related antioxidant gene expression is as follows: extracting RNA from the sample, synthesizing cDNA with the extracted RNA as template, detecting by using qPCR method, the primers used in the qPCR are for GPx gene and GCLC gene, and β-actin is used as an internal reference.

Images