Method for improving digestion and absorption efficiency and antibacterial immune components of maggots

Abstract

The invention relates to the technical field of medicinal breeding of special insects, in particular to a method for improving digestion and absorption efficiency and antibacterial immune components of maggots, and the method comprises the following steps: preparing a fish-hawthorn-astragalus membranaceus fermented feed; continuous three generations of directional stress screening are carried out, offspring is used as next generation provenance, and a target population is obtained; the three continuous generations of directional stress screening are performed according to the following modes: first-generation screening: feeding larvae with fermented feed, and applying high-protein stress; the larvae are fed with fermented feed added with astragaloside, low-temperature stress is applied, and individuals with the immune globulin IMPI content larger than or equal to 600 mu g / g are screened; and third-generation screening: feeding the larvae with fermented feed, and applying high-protein and low-temperature composite stress. The fish-hawthorn-astragalus membranaceus fermented feed is prepared in combination with continuous three-generation directional stress screening, so that the effects of activating intestinal protease of maggots to improve the digestion efficiency, promoting immune cell differentiation to accumulate immune components, enhancing intestinal flora regulation and enhancing the digestion and immune synthesis capability are achieved.

Description

Technical Field

[0001] This invention relates to the field of medicinal insect breeding technology, specifically a method for improving the digestion and absorption efficiency and antibacterial and immune components of the grain insect. Background Technology

[0002] In the field of medicinal insect farming, enhancing the nutritional and functional characteristics of the bark fly larvae (Goldfly larvae) has always been a key research and development focus. Early technological approaches primarily revolved around protein fortification of basic feeds, such as using fish by-products fermented by microorganisms to produce high-protein feed, directly promoting insect growth. With a deeper understanding of insect digestive physiology and immune mechanisms, researchers began exploring the introduction of medicinal and edible plant components into feed systems. For example, some researchers have added powdered hawthorn or astragalus root into feed in a simple form, hoping to utilize the organic acids in hawthorn to aid fat breakdown, or to use astragalus extracts to non-specifically stimulate the insect's immune system. These explorations mark a significant shift in the field, moving from providing basic nutrition to attempting targeted regulation of insect metabolic states.

[0003] Despite these challenges, existing methods still face several significant difficulties in practical application. First, the large protein molecules in traditional fish-based fermented feeds are often difficult for insects to efficiently break down and utilize, resulting in persistently low protein digestibility and wasted raw materials. Second, while functional ingredients such as hawthorn and astragalus are introduced, common, simple addition methods fail to fully release and transform their active substances, preventing these components from effectively complementing the feed matrix. Consequently, their activation of the insect's digestive enzyme system is limited, and their effect on enhancing immune function is less than ideal. More importantly, most existing farming models lack a coherent and targeted physiological adaptation mechanism, failing to stably translate the potential benefits of feed into functional enhancements within the insects. This ultimately leads to low and fluctuating accumulation levels of high-value components such as antimicrobial peptides and immunoglobulins in the insects. Therefore, how to establish a complete chain from feed improvement and digestion promotion to immune enhancement to achieve stable and efficient production of functional components remains a core unresolved issue in current technologies. Summary of the Invention

[0004] The purpose of this invention is to provide a fish-hawthorn-astragalus fermented feed, which activates the digestive enzyme system of the five grain insects through the active ingredients of traditional Chinese medicine (hawthorn acid and astragalus polysaccharide), promotes the decomposition of fish protein and the absorption of immune components, and enhances the content of antimicrobial peptides and immunoglobulins by combining 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 improving the digestibility and absorption efficiency and antibacterial and immune components of the grain worm, which is the larva of the big-headed golden fly, includes the following steps:

[0007] (1) Preparation of fish-hawthorn-astragalus fermented feed;

[0008] (2) Based on fermented feed, conduct three consecutive generations of targeted stress screening, and use the offspring as the breeding source for the next generation to obtain the target population; the three consecutive generations of targeted stress screening are carried out in the following manner:

[0009] First-generation screening: Larvae were fed fermented feed and subjected to high protein stress, and individuals with protease activity ≥150U / mg were selected.

[0010] Second-generation screening: Larvae were fed fermented feed supplemented with astragaloside A and subjected to low-temperature stress. Individuals with immunoglobulin IMPI content ≥600μg / g were selected.

[0011] Third-generation screening: Larvae were fed fermented feed and subjected to a combination of high protein and low temperature stress. Individuals with antimicrobial peptide cecropin ≥750μg / g and IMPI ≥800μg / g were selected.

[0012] Furthermore, in step (1), the preparation of the fish-hawthorn-astragalus fermented feed specifically includes the following steps:

[0013] S1. Raw material pretreatment: Take 60% fresh miscellaneous fish, 20% dried hawthorn, 15% dried astragalus, and 5% wheat bran by weight percentage; crush the dried hawthorn and pass it through a 40-mesh sieve, then decoct it at 80℃ for 2 hours, filter and take the supernatant; crush the dried astragalus and mix it evenly with the wheat bran to obtain an astragalus-wheat bran mixture; mince the fresh miscellaneous fish to a particle size ≤2mm to obtain fish paste;

[0014] S2. Mixed Fermentation: Fish paste, hawthorn supernatant, and astragalus-wheat bran mixture are mixed sequentially. 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation are added. The mixture is then placed in an anaerobic fermentation environment at 28℃ for 72 hours until the pH is ≤4.8 and stable for more than 12 hours. The fermentation product obtained contains ≥0.5% hawthorn acid, ≥1.2% astragalus polysaccharide, and ≥75% small peptides with a molecular weight ≤8kDa.

[0015] Furthermore, in step S2, the specific operation and monitoring process for mixed fermentation is as follows:

[0016] S21. Mixing operation: Place the fish paste in a stainless steel mixing tank, slowly add the hawthorn supernatant and stir at a low speed of 100 rpm, then add the astragalus-wheat bran mixture and continue stirring for 5 minutes until uniform, finally add 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation, seal the tank opening and transfer to an anaerobic fermentation environment.

[0017] S22. Fermentation monitoring: Samples are taken every 6 hours during fermentation, and the pH of the mixture is measured using a calibrated pH meter with an accuracy of ±0.01. The measured values ​​are recorded and a pH change curve is plotted until the pH is ≤4.8 and stabilizes for more than 12 hours.

[0018] Furthermore, the preparation process for each generation of breeding stock 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 a mixture of 85% fermented feed, 5% yeast extract, 5% glucose, and 5% vitamin solution by weight percentage. The vitamin solution contains 0.02% VC and 0.01% VE. After mixing, the moisture content is adjusted to 60%. A dark environment is maintained during the induction of egg laying. After the eggs are disinfected with ultraviolet light for 30 minutes, they are incubated for 10-12 hours at 28℃ and 70% humidity.

[0019] Furthermore, high protein stress is defined as feed crude protein content ≥45%, determined by the Kjeldahl method, applied continuously for 5 days, with equal amounts fed at fixed times each day; low temperature stress is defined as 6 hours of 20°C environmental treatment per day, with the rest of the time restored to 28°C, and temperature fluctuation ≤±1°C.

[0020] Furthermore, the amount of astragaloside A added to the feed used in the second-generation screening was 0.05%.

[0021] Furthermore, the storage conditions for fermented feed are as follows: sealed in food-grade polyethylene bags, placed in a cool, dry place with a temperature ≤10℃ and relative humidity ≤60%, protected from light and odors. Under these storage conditions, there is a specified shelf life. During this period, the pH, hawthorn acid, and astragalus polysaccharide content are checked regularly. When the pH >5.0 or the active ingredient decreases by ≥10%, use should be discontinued.

[0022] Furthermore, the processing procedure for the target population is as follows: after the larvae in the third instar are withheld from feeding for 6 hours, they are rinsed with low-temperature physiological saline to remove surface dirt, rinsed twice with running cold water, dried with low-temperature hot air until the moisture content is ≤8%, graded according to body length and color uniformity, and then vacuum-packed in food-grade aluminum foil composite bags.

[0023] Furthermore, in the first-generation screening, protease activity was detected using the Folin-phenol method, and enzyme activity was calculated using the following formula:

[0024]

[0025] Where μmol Tyr is the number of micromoles of tyrosine released, min is the reaction time in minutes, and mg protein is the number of milligrams of protein in the sample.

[0026] Furthermore, a method for improving the digestive and absorptive efficiency and antibacterial immune components of the grain insect, a domesticated grain insect population with protease activity ≥180U / mg, antimicrobial peptide cecropin ≥780μg / g, and IMPI ≥850μg / g, when continuously reproduced to the sixth generation, the coefficient of variation of the above indicators is <5%, and the traits are stably inherited.

[0027] The advantages of this invention compared to the prior art are:

[0028] 1. This invention prepares a fish-hawthorn-astragalus fermented feed containing hawthorn acid, and combines it with a first-generation high-protein stress targeted screening strategy to effectively activate the intestinal protease activity of the five-grain insect and upregulate its expression level. The small peptides with appropriate molecular weight contained in the fermented feed can further improve the nutrient absorption efficiency, ultimately achieving a digestive enhancement effect of increasing fish protein digestibility from 70% to 92% and feed utilization rate by 30%.

[0029] 2. This invention utilizes the nourishing and regulatory effects of astragalus polysaccharides in fermented feed, combined with second-generation fermented feed supplemented with astragaloside A and low-temperature stress screening, followed by third-generation compound stress screening to solidify immune-related traits. This significantly promotes the differentiation and maturation of immune cells in *Strombus haematomarginatus*, ultimately achieving a 40% increase in IMPI content and a 56% increase in antimicrobial peptide content. Simultaneously, it greatly enhances the resistance of *Strombus haematomarginatus* to Staphylococcus aureus and significantly improves the overall disease resistance of the population.

[0030] 3. This invention provides precise nutritional support through specialized fermented feed, combined with the optimization of population characteristics through three generations of targeted stress screening. This effectively regulates the composition of the gut microbiota of the grain insect, causing the expression level of digestive enzyme genes in the intestine to increase by two times. This achieves simultaneous enhancement of the grain insect's own digestive function and immune component synthesis ability, making it a natural biotransformation carrier with both high digestive efficiency and high immune component synthesis ability, thus greatly improving the practical application value of this grain insect population. Attached Figure Description

[0031] 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.

[0032] In the attached diagram:

[0033] Figure 1 This is a table of experimental data for a method in Example 1 to improve the digestibility and absorption efficiency of grain insects and enhance their antibacterial and immune components. Detailed Implementation

[0034] The following detailed description of the embodiments is used to exemplarily illustrate the principles of this application, but should not be used to limit the scope of this application. That is, the method of this application for improving the digestion and absorption efficiency and antibacterial immune components of grain insects is not limited to the described embodiments.

[0035] The present invention will be further described below with reference to embodiments.

[0036] Example 1

[0037] like Figure 1 As shown, a method for improving the digestibility and absorption efficiency and antibacterial and immune components of the grain worm (a type of fly larvae) is disclosed. The method includes the following steps:

[0038] (1) Preparation of fish-hawthorn-astragalus fermented feed;

[0039] (2) Based on fermented feed, conduct three consecutive generations of targeted stress screening, and use the offspring as the breeding source for the next generation to obtain the target population; the three consecutive generations of targeted stress screening are carried out in the following manner:

[0040] First-generation screening: Larvae were fed fermented feed and subjected to high protein stress, and individuals with protease activity ≥150U / mg were selected.

[0041] Second-generation screening: Larvae were fed fermented feed supplemented with astragaloside A and subjected to low-temperature stress. Individuals with immunoglobulin IMPI content ≥600μg / g were selected.

[0042] Third-generation screening: Larvae were fed fermented feed and subjected to a combination of high protein and low temperature stress. Individuals with antimicrobial peptide cecropin ≥750μg / g and IMPI ≥800μg / g were selected.

[0043] In a specific embodiment, this method was implemented using a fish-hawthorn-astragalus fermented feed combined with three generations of ecological domestication. First, a special fermented feed was prepared, followed by adult oviposition induction. An induction feed containing 85% fermented feed, 5% yeast extract, 5% glucose, and 5% vitamins was selected, with the moisture content adjusted to 60%. Adults were induced to lay eggs in concentrated quantities for 1 to 2 hours, maintaining a dark environment during the induction period. After the eggs were sterilized with ultraviolet light for 30 minutes, they were placed in an environment at 28°C and 70% relative humidity for incubation for 10 to 12 hours. For the first generation screening, 1st to 3rd instar larvae were fed the fermented feed, and high-protein stress was applied from the second day of the 1st instar for 5 consecutive days, with equal amounts fed at fixed times each day. Individuals with protease activity ≥150 U / mg were selected, totaling 2800 individuals. For the second generation screening, the recovered eggs from the first generation were hatched, and the larvae were fed fermented feed supplemented with astragaloside A. Low-temperature stress was applied for 6 hours daily during the 1st to 3rd instar stages, and individuals with IMPI content ≥600 μg / g were selected. The third-generation selection involved hatching eggs recovered from the second generation. The larvae were fed the original fermented feed and simultaneously subjected to a combination of high protein and low temperature stress. Individuals with antimicrobial peptides ≥750μg / g and IMPI ≥800μg / g were selected, ultimately yielding 1500 target individuals.

[0044] Furthermore, in step (1), the preparation of the fish-hawthorn-astragalus fermented feed specifically includes the following steps:

[0045] S1. Raw material pretreatment: Take 60% fresh miscellaneous fish, 20% dried hawthorn, 15% dried astragalus, and 5% wheat bran by weight percentage; crush the dried hawthorn and pass it through a 40-mesh sieve, then decoct it at 80℃ for 2 hours, filter and take the supernatant; crush the dried astragalus and mix it evenly with the wheat bran to obtain an astragalus-wheat bran mixture; mince the fresh miscellaneous fish to a particle size ≤2mm to obtain fish paste;

[0046] S2. Mixed Fermentation: Fish paste, hawthorn supernatant, and astragalus-wheat bran mixture are mixed sequentially. 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation are added. The mixture is then placed in an anaerobic fermentation environment at 28℃ for 72 hours until the pH is ≤4.8 and stable for more than 12 hours. The fermentation product obtained contains ≥0.5% hawthorn acid, ≥1.2% astragalus polysaccharide, and ≥75% small peptides with a molecular weight ≤8kDa.

[0047] In this specific embodiment, fermented feed was prepared strictly according to the percentage of raw materials. During raw material pretreatment, dried hawthorn was pulverized and passed through a 40-mesh sieve, then decocted at 80°C for 2 hours. The supernatant was then filtered and reserved. Dried astragalus was pulverized and thoroughly mixed with wheat bran. Fresh miscellaneous fish was minced to a particle size ≤2mm to obtain fish paste. In the mixed fermentation stage, the fish paste, hawthorn supernatant, and astragalus-wheat bran mixture were mixed sequentially. Then, 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation were added, and the mixture was placed in an anaerobic environment at 28°C for 72 hours. The pH value was continuously monitored during fermentation until it reached ≤4.8 and remained stable for more than 12 hours. After fermentation, the product indicators were tested, revealing an astragalus polysaccharide content of 1.5%, a hawthorn acid content of 0.6%, and a proportion of small peptides with a molecular weight ≤8kDa meeting the requirements.

[0048] Furthermore, in step S2, the specific operation and monitoring process for mixed fermentation is as follows:

[0049] S21. Mixing operation: Place the fish paste in a stainless steel mixing tank, slowly add the hawthorn supernatant and stir at a low speed of 100 rpm, then add the astragalus-wheat bran mixture and continue stirring for 5 minutes until uniform, finally add 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation, seal the tank opening and transfer to an anaerobic fermentation environment.

[0050] S22. Fermentation monitoring: Samples are taken every 6 hours during fermentation, and the pH of the mixture is measured using a calibrated pH meter with an accuracy of ±0.01. The measured values ​​are recorded and a pH change curve is plotted until the pH is ≤4.8 and stabilizes for more than 12 hours.

[0051] In a specific embodiment, the mixing operation was carried out in a stainless steel mixing tank. First, the fish paste was placed in the tank, and hawthorn supernatant was slowly added while stirring was started at 100 rpm. After adding the astragalus-wheat bran mixture, stirring continued for 5 minutes to ensure the mixture was homogeneous. Then, 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation were added, and the tank was immediately sealed and transferred to an anaerobic fermentation environment. During fermentation monitoring, samples were taken every 6 hours, and the pH value was measured using a calibrated pH meter with an accuracy of ±0.01. Each measurement was recorded in detail, and a pH change curve was plotted based on the recorded data. The pH trend was observed through the curve until the pH ≤ 4.8 and remained stable for more than 12 hours, confirming the fermentation endpoint.

[0052] Furthermore, the preparation process for each generation of breeding stock 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 a mixture of 85% fermented feed, 5% yeast extract, 5% glucose, and 5% vitamin solution by weight percentage. The vitamin solution contains 0.02% VC and 0.01% VE. After mixing, the moisture content is adjusted to 60%. A dark environment is maintained during the induction of egg laying. After the eggs are disinfected with ultraviolet light for 30 minutes, they are incubated for 10-12 hours at 28℃ and 70% humidity.

[0053] In a specific embodiment, the preparation of the breeding stock follows a standardized procedure. The inducing feed is prepared according to a specified mass percentage, ensuring the vitamin mixture contains 0.02% VC and 0.01% VE. After thorough mixing of all ingredients, the moisture content is adjusted to 60%, and the feed is prepared and used immediately. This inducing feed is used to induce adult insects to lay eggs in concentrated quantities for 1 to 2 hours. A dark environment is maintained during the induction process to increase egg-laying concentration. After egg-laying, the eggs are collected, disinfected with ultraviolet light for 30 minutes, and then placed in an environment at 28°C and 70% relative humidity for incubation. The incubation period is controlled to be 10 to 12 hours to ensure normal hatching of larvae.

[0054] Furthermore, high protein stress is defined as feed crude protein content ≥45%, determined by the Kjeldahl method, applied continuously for 5 days, with equal amounts fed at fixed times each day; low temperature stress is defined as 6 hours of 20°C environmental treatment per day, with the rest of the time restored to 28°C, and temperature fluctuation ≤±1°C.

[0055] In a specific embodiment, high-protein stress was achieved using a specialized feed. High-quality fishmeal was added to the basal fermented feed, and the crude protein content was ensured to be ≥45% by Kjeldahl method. Feeding was conducted in equal amounts at 08:00 and 18:00 daily, ensuring the larvae consumed the feed within one hour to prevent spoilage. The stress lasted for five consecutive days starting from the second day of the first instar, during which the larvae's feed intake and activity were observed daily. Low-temperature stress was conducted using a temperature-controlled incubator. From 09:00 to 15:00 daily, the incubator was moved to a 20°C environment, and the temperature was restored to 28°C for the rest of the time. The incubator temperature was recorded hourly to ensure temperature fluctuations were ≤±1°C. For combined stress, high-protein feed feeding and 6 hours of daily low-temperature treatment were implemented simultaneously until the end of the third instar.

[0056] Furthermore, the amount of astragaloside A added to the feed used in the second-generation screening was 0.05%.

[0057] In a specific embodiment, the feed used for the second-generation screening was supplemented with astragaloside A as required, with the addition amount precisely controlled at 0.05%. During feed formulation, astragaloside A was thoroughly mixed with the base fish-hawthorn-astragalus fermented feed to ensure a uniform distribution of astragaloside A in the feed. This feed was then used to feed the second-generation larvae, along with low-temperature stress treatment, ensuring an adequate feed supply during the rearing process. Once the larvae reached a suitable growth stage, their IMPI content was measured, and individuals with an IMPI content ≥600 μg / g were selected as the third-generation breeding stock.

[0058] Furthermore, the storage conditions for fermented feed are as follows: sealed in food-grade polyethylene bags, placed in a cool, dry place with a temperature ≤10℃ and relative humidity ≤60%, protected from light and odors. Under these storage conditions, there is a specified shelf life. During this period, the pH, hawthorn acid, and astragalus polysaccharide content are checked regularly. When the pH >5.0 or the active ingredient decreases by ≥10%, use should be discontinued.

[0059] In a specific embodiment, after the fermented feed is prepared, it is sealed and packaged in food-grade polyethylene bags. The storage environment should be a cool, dry place, with a temperature controlled at ≤10℃ and relative humidity at ≤60%, while ensuring protection from light and odors. Under these storage conditions, the feed can be stably preserved. During storage, samples are taken every 15 days, and the tests include pH value, hawthorn acid content, and astragalus polysaccharide content. Each test uses instruments with qualified accuracy. If the test shows pH > 5.0 or a decrease in active ingredients ≥ 10%, the batch of feed is considered invalid and its use should be immediately discontinued.

[0060] Furthermore, the processing procedure for the target population is as follows: after the larvae in the third instar are withheld from feeding for 6 hours, they are rinsed with low-temperature physiological saline to remove surface dirt, rinsed twice with running cold water, dried with low-temperature hot air until the moisture content is ≤8%, graded according to body length and color uniformity, and then vacuum-packed in food-grade aluminum foil composite bags.

[0061] In a specific embodiment, the processing of the target population was carried out according to standard procedures. Third-instar larvae were selected, withheld from feeding for 6 hours, then rinsed with low-temperature physiological saline to remove surface dirt, followed by two rinses with running cold water to remove residual dirt and feed residue. After cleaning, they were dried using a low-temperature hot air drying method, with the drying temperature controlled at 45℃ and the air velocity at 1.5m / s. The moisture content was tested according to the oven drying method GB 5009.3 until it was ≤8%. After drying, the larvae were classified into premium, first-grade, and second-grade according to body length ≥12mm and color uniformity. After grading, they were vacuum-packed in food-grade aluminum foil composite bags, and the batch number, production date, and shelf life were labeled after packaging.

[0062] Furthermore, in the first-generation screening, protease activity was detected using the Folin-phenol method, and enzyme activity was calculated using the following formula:

[0063]

[0064] Where μmol Tyr is the number of micromoles of tyrosine released, min is the reaction time in minutes, and mg protein is the number of milligrams of protein in the sample.

[0065] In a specific embodiment, the Folin-phenol method was strictly used for protease activity detection during the first-generation screening. Before detection, samples were prepared by adding pre-cooled physiological saline to late-stage larvae at a weight-to-volume ratio of 1:9, homogenizing, and centrifuging at 10,000 rpm for 10 minutes at 4°C. The supernatant was used as the test sample. The reaction system consisted of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, and 1 mM casein substrate. After adding the sample to the reaction system, the reaction was carried out at 37°C for 10 minutes. The reaction was terminated by adding 10% TCA, followed by centrifugation after 10 minutes on ice. The supernatant was then collected. The amount of tyrosine released was determined using the Folin-phenol method, and the enzyme activity was calculated using the formula. Accurate calculation of the micromoles of released tyrosine, reaction time (in minutes), and protein content (in milligrams) in the sample was crucial to ensure accurate results.

[0066] Furthermore, a method for improving the digestive and absorptive efficiency and antibacterial immune components of the grain insect, a domesticated grain insect population with protease activity ≥180U / mg, antimicrobial peptide cecropin ≥780μg / g, and IMPI ≥850μg / g, when continuously reproduced to the sixth generation, the coefficient of variation of the above indicators is <5%, and the traits are stably inherited.

[0067] In a specific embodiment, a target grain insect population was obtained through domestication. Testing showed that this population had a protease activity of 180±10 U / mg, an antimicrobial peptide cecropin content of 780±25 μg / g, and an IMPI content of 850±30 μg / g. To verify the stability of the traits, this population was used as a parent for three consecutive generations: the fourth, fifth, and sixth generations. Each generation followed the same domestication procedure, and the three key indicators were tested at the end of the third instar. The results showed that the fourth generation had a protease activity of 178±12 U / mg, an antimicrobial peptide content of 775±28 μg / g, and an IMPI content of 845±32 μg / g; the fifth generation had a protease activity of 179±11 U / mg, an antimicrobial peptide content of 778±27 μg / g, and an IMPI content of 848±29 μg / g; and the sixth generation had a protease activity of 181±9 U / mg, an antimicrobial peptide content of 782±24 μg / g, and an IMPI content of 852±31 μg / g. The coefficients of variation for all three indicators were <5% across three consecutive generations, indicating stable inheritance of the traits. Furthermore, SDS-PAGE analysis showed that the antimicrobial peptide bands in this population were clear, with a molecular weight of 3 to 5 kDa.

[0068] 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.

[0069] 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 digestibility and absorption efficiency and antibacterial and immune components of grain weevils, characterized in that, The five-grain worm is the larva of the big-headed goldfly, and the method includes the following steps: (1) Preparation of fish-hawthorn-astragalus fermented feed; (2) Based on fermented feed, conduct three consecutive generations of targeted stress screening, and use the offspring as the breeding source for the next generation to obtain the target population; the three consecutive generations of targeted stress screening are carried out in the following manner: First-generation screening: Larvae were fed the fermented feed and subjected to high protein stress, and individuals with protease activity ≥150U / mg were screened. Second-generation screening: Larvae were fed the fermented feed supplemented with astragaloside A and subjected to low-temperature stress to screen individuals with an immunoglobulin IMPI content ≥600μg / g; Third-generation screening: Larvae were fed the fermented feed and subjected to a combination of high protein and low temperature stress. Individuals with antimicrobial peptide cecropin ≥750μg / g and IMPI ≥800μg / g were selected.

2. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain worms according to claim 1, characterized in that: In step (1), the preparation of the fish-hawthorn-astragalus fermented feed specifically includes the following steps: S1. Raw material pretreatment: Take 60% fresh miscellaneous fish, 20% dried hawthorn, 15% dried astragalus, and 5% wheat bran by weight percentage; crush the dried hawthorn and pass it through a 40-mesh sieve, then decoct it at 80℃ for 2 hours, filter and take the supernatant; crush the dried astragalus and mix it evenly with the wheat bran to obtain an astragalus-wheat bran mixture; mince the fresh miscellaneous fish to a particle size ≤2mm to obtain fish paste; S2. Mixed fermentation: The fish paste, hawthorn supernatant, and astragalus-wheat bran mixture are mixed sequentially, and 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation are added. The mixture is then placed in an anaerobic fermentation environment at 28℃ for 72 hours until the pH is ≤4.8 and stable for more than 12 hours. The fermentation product obtained contains ≥0.5% hawthorn acid, ≥1.2% astragalus polysaccharide, and ≥75% small peptides with a molecular weight ≤8kDa.

3. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain worms according to claim 2, characterized in that: In step S2, the specific operation and monitoring process of the mixed fermentation is as follows: S21. Mixing operation: Place the fish paste in a stainless steel mixing tank, slowly add hawthorn supernatant and stir at a low speed of 100 rpm, then add astragalus-wheat bran mixture and continue stirring for 5 minutes until uniform, finally add 3% lactic acid bacteria preparation and 2% Bacillus subtilis preparation, seal the tank opening and transfer to an anaerobic fermentation environment. S22. Fermentation monitoring: Samples are taken every 6 hours during fermentation, and the pH of the mixture is measured using a calibrated pH meter with an accuracy of ±0.

01. The measured values ​​are recorded and a pH change curve is plotted until the pH is ≤4.8 and stabilizes for more than 12 hours.

4. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain worms according to claim 3, characterized in that: The preparation process for each generation of breeding stock is as follows: Adult insects are induced to lay eggs in a concentrated manner for 1-2 hours using an inducing feed. The inducing feed consists of 85% fermented feed, 5% yeast extract, 5% glucose, and 5% vitamin mixture by weight percentage. The vitamin mixture contains 0.02% VC and 0.01% VE. After mixing, the moisture content is adjusted to 60%. A dark environment is maintained during the induction of egg laying. After the eggs are disinfected with ultraviolet light for 30 minutes, they are incubated at 28℃ and 70% humidity for 10-12 hours.

5. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain weevils according to claim 4, characterized in that: The high protein stress refers to feed with a crude protein content ≥45%, determined by the Kjeldahl nitrogen determination method, applied continuously for 5 days, with equal amounts fed at fixed times each day; the low temperature stress refers to environmental treatment at 20℃ for 6 hours each day, followed by restoration to 28℃ for the rest of the time, with temperature fluctuations ≤±1℃.

6. The method for improving the digestibility and absorption efficiency and antibacterial immune components of grain weevils according to claim 5, characterized in that: The amount of astragaloside A added to the feed used in the second-generation screening was 0.05%.

7. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain weevils according to claim 6, characterized in that: The storage conditions for the fermented feed are as follows: it is sealed in a food-grade polyethylene bag and placed in a cool, dry place with a temperature ≤10℃ and relative humidity ≤60%, away from light and odors. Under these storage conditions, it has a specified shelf life. During this period, the pH, hawthorn acid and astragalus polysaccharide content are checked regularly. When the pH >5.0 or the active ingredient decreases by ≥10%, it is no longer used.

8. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain worms according to claim 7, characterized in that: The processing procedure for the target population is as follows: after the larvae in the third instar are withheld from feeding for 6 hours, they are rinsed with low-temperature physiological saline to remove surface dirt, rinsed twice with running cold water, dried with low-temperature hot air until the moisture content is ≤8%, graded according to body length and color uniformity, and then vacuum-packed in food-grade aluminum foil composite bags.

9. The method for improving the digestibility and absorption efficiency and antibacterial and immune components of grain worms according to claim 8, characterized in that: In the first-generation screening, protease activity was detected using the Folin-phenol method, and enzyme activity was calculated using the following formula: Where μmol Tyr is the number of micromoles of tyrosine released, min is the reaction time in minutes, and mg protein is the number of milligrams of protein in the sample.

10. A population of *Strombus spp.* obtained by domestication using the method described in any one of claims 1-9 for improving the digestibility and absorption efficiency and antibacterial immune components of *Strombus spp.*, characterized in that... The population exhibits protease activity ≥180 U / mg, antimicrobial peptide cecropin ≥780 μg / g, and IMPI ≥850 μg / g. When continuously propagated to the sixth generation, the coefficient of variation for the above indicators is <5%, indicating that the traits are stably inherited.

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