Application of canthaxanthin extract and feed additive containing canthaxanthin extract in silkworm breeding

Abstract

The invention relates to the technical field of biological medicines, in particular to application of canthaxanthin extract and a feed additive containing the canthaxanthin extract in silkworm breeding, and finds that cocoon shell amount, whole cocoon amount and pupa weight of silkworms can be remarkably increased and egg laying amount of single moths can be increased by adding canthaxanthin extract to silkworms for the first time. Meanwhile, the silkworm variety with functional carotenoid binding protein can also be induced to produce orange red natural colored cocoons. The invention further provides a compound feed additive containing the canthaxanthin extract and the sericin-derived small peptide and / or gamma-aminobutyric acid. Experiments prove that the canthaxanthin extract and the sericin-derived small peptide have a synergistic effect in the aspect of increasing the cocoon shell amount. According to the technical scheme, on the premise that normal growth and development of silkworms are not affected, the multiple purposes of yield increasing, propagation promoting and color increasing are achieved at the same time, and the method has good application prospects and industrial value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of anastellarin extract and a feed additive containing the same in silkworm breeding. BACKGROUND

[0002] Natural colored silk is attracting much attention in the field of high-end textiles, biomaterials and functional clothing due to its environmental friendliness, color durability and no need for post-dyeing and finishing. At present, the main approaches to produce colored silk include variety selection, genetic engineering, physical dyeing and feed supplementation with pigments. However, these methods still have significant limitations in terms of safety, pigment source, production efficiency and comprehensive effects on the physiological characteristics of silkworms.

[0003] Chinese patent document CN101285215B discloses a method for preparing yellow silk, which involves feeding silkworms with acid yellow 73 (a synthetic dye) in combination with antibiotics and specific additives to improve the uniformity and color fastness of the colored silk. This method demonstrates the feasibility of regulating cocoon color through feed supplementation, and the pigment used belongs to the category of synthetic dyes.

[0004] Chinese patent document CN1430904A discloses a process for producing colored cocoon silk by feeding silkworms with multiple dye pigments (including acid, basic and reactive types). This technology further broadens the range of cocoon colors that can be obtained by feed supplementation, and points out that it is applicable to various silkworm varieties. The pigment system relied on by this process is also based on synthetic dyes.

[0005] Chinese patent document CN107318790B provides a different technical idea, which is to breed silkworm varieties with yellow-green cocoon limiting traits through genetic breeding, thereby achieving natural separation of male and female cocoons. This method can obtain genetically stable natural colored cocoons, but it involves a long hybridization and selection process, and the cocoon color is determined by the genetic background, making it difficult to flexibly adjust or quickly expand to other color systems.

[0006] In summary, in the prior art, although the method of feed supplementation with synthetic dyes can flexibly regulate cocoon color to some extent, the types of pigments used and their long-term biological effects need further research. The method of obtaining natural colored cocoons through genetic selection avoids the addition of exogenous substances, but the breeding cycle is long and the color expansion is limited. In addition, the above technical solutions mainly focus on the generation and control of cocoon color itself, and usually do not systematically explore and utilize whether and how the feed substances can simultaneously improve key economic traits of silkworms (such as egg laying amount, cocoon layer amount, whole cocoon amount, etc.). Therefore, how to safely and effectively introduce new colors in silkworm breeding and simultaneously optimize the production performance of silkworms is still a technical problem worth exploring in the field. SUMMARY

[0007] To solve the above technical problems, the present application aims to provide the application of canthaxanthin extract and feed additive containing the same in silkworm breeding.

[0008] To achieve the above technical effects, the present application adopts the following technical solutions: In the first aspect of the present application, the application of canthaxanthin extract in silkworm breeding is provided.

[0009] As an embodiment of the present application, the application is to increase the egg-laying amount of silkworm by feeding canthaxanthin extract to silkworm.

[0010] As another embodiment of the present application, the application is to produce orange-red cocoon by feeding canthaxanthin extract to silkworm.

[0011] As a preferred embodiment of the present application, the application is to increase the egg-laying amount of silkworm and produce orange-red cocoon at the same time by feeding canthaxanthin extract to silkworm.

[0012] In the second aspect of the present application, a feed additive for silkworm breeding is provided.

[0013] As an embodiment of the present application, the feed additive comprises canthaxanthin extract.

[0014] As a preferred embodiment of the feed additive of the present application, the feed additive further comprises any one or a mixture of both of sericin-derived small peptides and / or gamma-aminobutyric acid. By compounding canthaxanthin extract with sericin-derived small peptides and / or gamma-aminobutyric acid, a synergistic effect can be produced, further improving the economic traits of silkworm.

[0015] As a further preferred embodiment of the feed additive of the present application, the sericin-derived small peptides are prepared by the following steps: (a) The silk fibroin or silk fibroin-rich silk processing by-products are subjected to preliminary alkaline treatment under the following conditions: pH 9.0-11.0, temperature 50℃-70℃, and time 0.5-2 hours; (b) The product of step (a) is adjusted to neutral, and a protease is added for enzymatic hydrolysis, wherein the protease is alkaline protease or complex protease, the enzymatic hydrolysis temperature is 45℃-55℃, and the time is 3-6 hours; (c) The enzymatic hydrolysate is inactivated, filtered, concentrated, and dried to obtain sericin-derived small peptides with a molecular weight less than 3000 Da.

[0016] The preparation method is simple in process and controllable in cost, uses sericin or a silk processing byproduct as a raw material, and realizes high-value utilization of a sericulture byproduct.

[0017] As a further preferred embodiment of the feed additive of the present application, the weight ratio of the canthaxanthin extract, the sericin-derived small peptide and the gamma-aminobutyric acid is 1:(0.03-1):(0.03-1).

[0018] In one specific embodiment of the present application, a ratio of 250 mg of canthaxanthin extract, 15 mg of sericin-derived small peptide and 15 mg of gamma-aminobutyric acid (corresponding to a weight ratio of 1:0.06:0.06) is used for compounding, and excellent technical effects are achieved.

[0019] As another preferred embodiment of the feed additive of the present application, the dosage form is any one of a solid preparation or a liquid preparation, and further preferably, the solid preparation includes but is not limited to a powder, a granule, a premix and the like; and the liquid preparation includes but is not limited to a solution, a suspension, an emulsion and the like, and a person skilled in the art can select a suitable dosage form according to actual production needs.

[0020] In a third aspect of the present application, the use of the feed additive of any one of the above in the preparation of a feed for silkworm feeding is provided, and by adding the feed additive of the present application in the feed preparation process, a feed for feeding with specific functions can be conveniently produced.

[0021] As a preferred embodiment of the application, the application concentration of the additive in the feed is: the application amount of 100-300 mg of canthaxanthin extract per 100 g of feed.

[0022] In one specific embodiment of the present application, the application concentration of 250 mg of canthaxanthin extract per 100 g of feed is used for feeding, and the results show that this dose is safe and effective, and can significantly improve the economic traits of silkworms. The application concentration range can be adjusted according to actual breeding needs, and the technical effects of the present application can be achieved.

[0023] As a preferred embodiment of the application, the feeding stage of the feed is the whole 5th instar period or the middle and late 5th instar period of silkworms. In one specific embodiment of the present application, the feeding is continuously started from the 1st day of the 5th instar to cocooning, and the whole 5th instar feeding is achieved, and good effects are achieved. A person skilled in the art can select a specific feeding time according to breeding practice.

[0024] In the fourth aspect of the present application, the feed additive described in any of the above is applied to improve the economic traits of the silkworm or to produce orange-red cocoon. As an embodiment of the present application, the economic traits are one or more of the increased egg laying amount, increased cocoon layer amount, increased whole cocoon amount, and increased pupa weight. The present application can simultaneously achieve the improvement of the above-mentioned multiple economic traits by feeding the silkworm with the canthaxanthin extract or the compound feed additive thereof, thereby significantly improving the overall benefit of silkworm breeding.

[0025] Compared with the prior art, the present application has the following beneficial effects: First, the present application first discovers and confirms the multiple effects of canthaxanthin extract in silkworm breeding. The experimental results show that by feeding the silkworm with canthaxanthin extract, the cocoon layer amount, whole cocoon amount and pupa weight of the silkworm can be significantly improved at a safe and non-toxic dose, thereby promoting the accumulation of cocoon silk yield and biomass; at the same time, the single moth egg laying amount of the silkworm can be significantly improved, and the reproductive performance can be improved; in addition, for silkworm varieties with functional carotenoid binding protein, orange-red natural colored cocoon with bright and uniform color can also be induced. The present application achieves the dual technical effects of simultaneously improving the economic traits of the silkworm and regulating the cocoon color without affecting the normal growth and development of the silkworm, thereby overcoming the single function limitation of the prior art.

[0026] Second, the present application further develops a compound feed additive containing canthaxanthin extract, silk sericin-derived small peptides and / or gamma-aminobutyric acid, and verifies the interaction of each component through systematic synergistic effect research. The experiment proves that canthaxanthin extract and silk sericin-derived small peptides have a significant synergistic effect on improving the cocoon layer amount of the silkworm, and the effect of the two components is much better than the simple addition of the effects of the two components used alone; on the basis of the synergistic effect of canthaxanthin extract and silk sericin-derived small peptides, the ternary compound additive composed of gamma-aminobutyric acid further improves the cocoon layer amount and egg laying amount, which are two key economic indicators, and the effect is significantly better than that of the binary combination. This finding provides a scientific basis and technical solution for the development of efficient and multifunctional silkworm breeding additives.

[0027] Third, the preparation method of the silk sericin-derived small peptides provided by the present application has the advantages of simple process and controllable cost. The silk sericin or silk processing by-products is used as raw material, and the small peptide product with a molecular weight of less than 3000 Da and easy to be absorbed and utilized by the silkworm is prepared by combining alkali treatment and enzymolysis. This technical route opens up a new way for the high-value utilization of mulberry and silk by-products, and helps to improve the overall economic benefit and sustainable development ability of the silk industry.

[0028] Fourthly, the canthaxanthin extract and the compound feed additive provided by the application have simple and easy application mode, only need to be sprayed on the surface of mulberry leaves at the 5th instar stage after being prepared into a solution, and can be applied in production practice without changing the existing breeding process and facilities, and has good industrial applicability and conversion prospect.

[0029] In summary, the application provides a technical solution capable of realizing the synergistic improvement of multiple targets of increasing production, promoting breeding and increasing color of silkworm, and has made significant technical progress in improving cocoon and silk yield, improving reproductive performance and developing new natural colored cocoon and silk, and has high industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The effect diagram of canthaxanthin extract feeding on cocoon color and silk gland color of different varieties of silkworm provided by an embodiment of the application; Among them, Figure 1 a is the cocoon and silk gland color comparison of the control group (CK group) and the canthaxanthin extract group (CAN group) of the golden cocoon variety; Figure 1 b is the cocoon and silk gland color comparison of the control group (CK group) and the canthaxanthin extract group (CAN group) of the white cocoon variety. DETAILED DESCRIPTION

[0031] The embodiments of the technical solution of the application will be described in detail below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the application, and therefore only serve as examples, and cannot limit the protection scope of the application.

[0032] Those skilled in the art should understand that the application can also be implemented without certain specific details. In some embodiments, methods, means, apparatus and steps familiar to those skilled in the art are not described in detail, in order to highlight the main idea of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. Unless otherwise specified, the units used in the specification are international standard units, and the numerical values and numerical ranges appearing in the application should be understood to include the systematic errors inevitable in industrial production.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents or instruments used are not marked with the manufacturer, and are reagents and materials that can be obtained from commercial channels; the specific conditions are not marked in the examples, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer, at the same time, the source of the raw materials used in the application is not limited, and the raw materials used in the application are ordinary commercial products in the technical field unless otherwise specified.

[0034] 1. Experimental Objective This systematic review evaluated the differential effects of supplementing the feed with canthaxanthin extract on the growth, development, silk yield, reproductive performance, and cocoon color of silkworms with golden cocoons containing functional carotenoid-binding protein (CBP) and white cocoons without functional CBP.

[0035] 2. Materials and Methods 2.1 Experimental Materials Canthaxanthin extract: derived from microbial fermentation, purchased from Shaanxi Panier Biotechnology Co., Ltd. (canthaxanthin content is 10%).

[0036] Silkworms used in the experiment: White cocoon variety (CBP defect type): Autumn White variety.

[0037] Golden Cocoon Variety (CBP Functional Type): XS17 Variety.

[0038] Basic feed: Uncontaminated fresh mulberry leaves.

[0039] Solvent: Pure water (used to dissolve and dilute canthaxanthin extract).

[0040] 2.2 Experimental Materials and Methods A completely randomized block design was used, with two treatment groups for each silkworm variety: Canthaxanthin extract experimental group (CAN group): fed mulberry leaves containing canthaxanthin extract.

[0041] Blank control group (CK group): fed with an equal amount of mulberry leaves containing pure water.

[0042] Each treatment group had 3 replicates, with 80 silkworms in their 5th instar in each replicate, for a total of 240 larvae per group.

[0043] 2.3 Feeding Treatment Accurately weigh the canthaxanthin extract and dissolve it in pure water to prepare a 10 mg / mL canthaxanthin extract supplementation solution. Before feeding, accurately measure 25 mL of this solution and spray it evenly on both sides of 100 g of fresh mulberry leaves using a small sprayer, then air dry. The calculated canthaxanthin extract content in the treated mulberry leaves was 250 mg / 100 g fresh leaves (i.e., 250 mg of canthaxanthin extract per 100 g of fresh mulberry leaves, with a canthaxanthin content of 10%). The control group's mulberry leaves were sprayed with an equal volume (25 mL) of pure water. Starting from day 1 of the 5th instar (referred to as L5D1), the treated mulberry leaves were fed daily at fixed times and in fixed quantities until all silkworms had spun cocoons, achieving continuous feeding throughout the 5th instar period.

[0044] 2.4 Observation Indicators and Measurement Methods Growth index and safety observation: Before feeding every morning, all surviving silkworms in each replicate were collectively weighed, the total weight was recorded, and the average single body weight (g) was calculated. At the same time, the death, abnormal behavior and poisoning symptoms of silkworms were observed and recorded daily.

[0045] Cocoon quality trait determination: After the silkworms were cocooned and pupated, 10 normal female cocoons and 10 male cocoons were randomly selected from each replicate on the 7th day after pupation. The total cocoon weight of each cocoon was weighed. Then the cocoon shell was cut open, and the pupa was taken out, and the pupa weight and cocoon layer weight were weighed. The cocoon layer rate was calculated according to the formula: cocoon layer rate (%) = (cocoon layer weight / total cocoon weight) x 100%.

[0046] Reproductive performance determination: After the silkworm moths emerged, male and female pairing was performed, and then the female moths after pairing were placed in a silkworm box covered with egg laying paper. All the eggs laid within 24 hours were collected, and the single moth egg laying amount was accurately counted.

[0047] Cocoon color phenotype observation and recording: All experimental groups of silkworm cocoons were observed, compared and photographed under standard D65 light source box.

[0048] 2.5 Data processing and analysis All experimental data were expressed as "mean ± standard deviation". Two-way ANOVA was performed using SPSS 26.0 to analyze the main effects of varieties and treatments and their interactions. If the interaction is significant or the main effect is significant, Tukey HSD method is used for multiple comparisons, and the significance level is set as P<0.05.

[0049] 3. Results and analysis 3.1 Effect of ocular extract supplement on the growth performance and safety of silkworms During the entire 5th instar feeding period, all treatment groups of silkworms showed normal performance, and no poisoning phenomena such as refusal to eat, vomiting, convulsions or mass death were observed, indicating that feeding ocular extract at a dose of 250 mg / 100g of feed was safe for silkworms. The body weight growth dynamics of two varieties of silkworms at each age in the 5th instar are shown in Table 1.

[0050] There was no significant difference between the CAN group and the CK group of the same variety and the same age (P > 0.05) by independent sample t-test. The weight gain of the golden cocoon variety slowed down during L5D3 to L5D4, which was the normal physiological characteristics of this variety at this development stage. The experimental results showed that there was no significant difference in the average weight of the CAN group and the CK group of either white cocoon variety or golden cocoon variety at any measured age. This indicates that, under the premise of safety, the addition of canthaxanthin extract to the diet does not significantly interfere with the normal growth and development process of the silkworm, neither showing toxic inhibition nor abnormal growth stimulation, providing a basis for its subsequent positive effects on cocoon quality and reproductive performance.

[0051] 3.2 Improvement of cocoon quality traits by canthaxanthin extract feeding To explore the effect of canthaxanthin extract on the economic traits of silkworm, 10 male and female cocoons were randomly selected from each replicate on the 7th day after pupation, and the whole cocoon weight, cocoon layer weight, pupa weight and cocoon layer rate were measured and calculated, as shown in Table 2.

[0052] The above experimental results show that the addition of canthaxanthin extract to the diet has a positive effect on the cocoon quality traits of both silkworm varieties.

[0053] For white cocoon varieties: compared with the CK group, the whole cocoon weight, cocoon layer weight and pupa weight of the CAN group of female and male silkworms showed significant increase (P < 0.05). Among them, the cocoon layer weight, which has the highest economic value, increased from 0.22 g to 0.26 g in female silkworms, with an increase of 18.2%; in male silkworms, it increased from 0.23 g to 0.26 g, with an increase of 13.0%. The cocoon layer rate of female silkworms also increased significantly. This indicates that canthaxanthin extract promotes the synthesis and deposition of nutrients to silk protein.

[0054] For golden cocoon varieties: similarly, the cocoon quality indicators of the CAN group were significantly better than those of the CK group (P < 0.05). The cocoon layer weight of male silkworms increased from 0.42 g to 0.45 g, and the pupa weight (an important indicator of biomass accumulation) of female silkworms increased from 1.32 g to 1.40 g.

[0055] The results show that the addition of canthaxanthin extract can effectively promote the conversion and accumulation of nutrients in silkworms, significantly increase the cocoon silk yield and overall biomass, and this effect is independent of the cocoon color type of the variety (i.e. the function of CBP), indicating that its mechanism of action may go beyond simple pigment transport, and it may have a positive regulatory effect on the overall physiological metabolism of silkworms.

[0056] 3.3 Promotion of silkworm reproductive performance by canthaxanthin extract feeding After the silkworm moths emerged, the number of eggs laid by a single moth was counted to evaluate the effect of canthaxanthin extract on the reproductive performance of silkworms. The results are shown in Table 3.

[0057] The above experimental results show that adding canthaxanthin extract to the diet significantly improved the reproductive capacity of the two silkworm varieties.

[0058] Specifically, the average egg production per moth in the white cocoon variety CAN group increased significantly by 4.9% compared to the CK group (P<0.05); the increase was even more pronounced in the golden cocoon variety CAN group, with a significant increase of 8.6% compared to the CK group (P<0.01). This result indicates that canthaxanthin extract not only improves silk production traits in silkworms but also significantly enhances their crucial reproductive trait of egg production. This may be related to its role as a strong antioxidant, which can scavenge reactive oxygen species accumulated during reproductive cell development, protecting oocytes from oxidative damage and thus increasing egg quantity and quality. This discovery greatly expands the application value of canthaxanthin extract in silkworm breeding and cocoon production.

[0059] 3.4 The regulatory effect of canthaxanthin extract on silkworm cocoon color 3.4 The regulatory effect of canthaxanthin extract on silkworm cocoon color The colors of silkworm cocoons in different treatment groups were observed and recorded, and the results are as follows: Figure 1 As shown.

[0060] The experimental results show that the cocoon color variation has significant variety specificity: like Figure 1 As shown in Figure a, for the golden cocoon variety, the cocoons spun in the CK group were uniformly golden yellow, with the silk glands in the middle being pale yellow. In contrast, the cocoons spun in the CAN group underwent a fundamental color change, exhibiting a bright, full, and uniform orange-red color. The color depth and saturation were significantly higher than the control golden cocoons, and the silk glands in the middle were clearly stained orange-red. This directly confirms that the canthaxanthin extract was effectively absorbed, transported, and deposited in the silk fibroin of the variety with functional CBP.

[0061] like Figure 1 As shown in b, for the white cocoon variety, the cocoons spun by both the CK and CAN groups were white, with no color difference visible to the naked eye. The central silk glands of the larvae in both groups remained colorless and transparent. This confirms that the absorption, transport, and deposition of canthaxanthin extract in the silkworm's body depends on the presence of functional carotenoid-binding proteins (CBPs). For the golden cocoon variety with functional CBPs, canthaxanthin extract can serve as a highly efficient exogenous pigment for the production of novel, high-value-added orange-red natural colored cocoons.

[0062] In summary, the results of Example 1 show that feeding silkworms with canthaxanthin extract (the content of canthaxanthin in the modified canthaxanthin extract is 10%) at a dose of 250 mg / 100 g of feed has no adverse effects on the growth and development of silkworms during the entire five instar feeding process, proving that the additive dose has good safety. In terms of cocoon quality traits, feeding canthaxanthin extract significantly increases the total cocoon weight, cocoon layer weight, and pupa weight of white cocoon and golden cocoon varieties, indicating that it has a positive promoting effect on silk yield and biomass accumulation. In terms of reproductive performance, the egg production of individual silkworms of both varieties is significantly improved after feeding canthaxanthin extract, indicating that it also has a positive impact on the reproductive capacity of silkworms. In terms of cocoon color regulation, for golden cocoon varieties with functional carotenoid-binding proteins (CBP), feeding canthaxanthin extract successfully induces the production of bright orange-red cocoon; while for white cocoon varieties with CBP defects, there is no change in cocoon color. In summary, canthaxanthin extract has dual functions of improving economic traits and regulating cocoon color in silkworm breeding, and has good application prospects.

[0063] Example 2 1. Purpose of the experiment The purpose of this embodiment is to provide a preparation method of a sericin-derived small peptide and a preparation and application of a compound feed additive containing the sericin-derived small peptide, and on the basis of Example 1, to explore the effect of the combination of canthaxanthin extract, sericin-derived small peptide, and gamma-aminobutyric acid (GABA).

[0064] 2. Materials and methods 2.1 Experimental materials Canthaxanthin extract: same as Example 1.

[0065] The preparation method of the sericin-derived small peptide includes the following steps: Alkaline pretreatment: take 100 grams of sericin protein powder (purchased from Jiangsu Yihao Tian Biotechnology Co., Ltd.) and add 1 liter of deionized water, stirring to disperse. Adjust the pH of the suspension to 10.0 with 1 M NaOH solution and place it in a 60°C constant temperature water bath, continuously stirring for 1 hour.

[0066] Enzymolysis: cool the above-mentioned alkali-treated mixture to 50°C, adjust the pH to 7.0 ± 0.2 with 1 M HCl solution. Add alkaline protease (Alcalase, enzyme addition amount is 1% of the mass of substrate sericin protein powder), and gently shake the enzyme in a 50°C constant temperature water bath shaker for 4 hours.

[0067] Post-treatment and purification: after the end of enzymolysis, the mixture was heated in a 95℃ water bath for 10 minutes to completely inactivate the enzyme. After cooling, it was first coarsely filtered through a 200-mesh screen and then separated by an ultrafiltration membrane system with a molecular weight cutoff of 3000 Da, and the filtrate (mainly containing small peptides with a molecular weight <3000 Da) was collected. The filtrate was concentrated to 1 / 5 of the original volume at 60℃ using a rotary evaporator, and finally vacuum freeze-dried to obtain white to light yellow powdery silk protein-derived small peptides.

[0068] Gamma-aminobutyric acid (GABA): purity ≥ 99%, commercially available.

[0069] Test silkworms: select the golden cocoon variety (XS17) with significant effect of adding ommochrome in Example 1.

[0070] Basic feed: uncontaminated fresh mulberry leaves.

[0071] 2.2 Experimental design A single-factor completely randomized design was adopted, and a total of 8 treatment groups were set up, with 3 replicates in each group and 80 5th instar silkworms in each replicate. The specific treatments of each group are shown in Table 4. The application methods of all additives were the same as in Example 1, i.e., they were dissolved in pure water and uniformly sprayed on the surface of mulberry leaves, starting from the 1st day of the 5th instar and continuously added until cocooning. Among them, the basic addition concentration of ommochrome extract was fixed at 250 mg / 100g of feed (same as the effective dose in Example 1), and other components were used at a dose of 15 mg / 100g of feed for single use or combination.

[0072] 2.3 Observation index The cocoon layer amount and egg laying amount, which best reflect the economic value, were mainly observed as the core index for evaluating the synergistic effect. At the same time, the growth and development of silkworms (body weight, mortality) and cocoon color phenotype were recorded. The determination method was the same as in Example 1.

[0073] 2.4 Synergistic effect evaluation method The Bliss independence model was used to evaluate the synergistic effect of binary combination and ternary combination. This model believes that the theoretical expected promotion rate of two independently acting compounds used in combination is calculated as follows: Observed promotion rate (P obs ) = (T treatment -T CK )) / T CK )×100%(T treatment -T CK )) / T CK )×100%, where T is the value of the measured index (such as cocoon layer amount).

[0074] Theoretical expected promotion rate (Pexp )=P A +P B -(P A ×P B ) / 100, wherein P A and P B are the measured promotion rates of A, B two single agent groups (or binary groups) (expressed in percentage, need to be divided by 100, or converted to percentage after decimal calculation).

[0075] For the ternary combination group (CAN+SP+GABA), it is regarded as the combined effect of canthaxanthin extract (CAN) and binary combination (SP+GABA), the theoretical expected promotion rate is calculated and compared with the measured value to evaluate the effect of ternary combination.

[0076] The judgment criteria for synergistic effect are: if (P obs -P exp )>10%, and the difference is significant after statistical analysis (such as t-test), it is considered to have synergistic effect; if between-10%~10%, it is simple additive effect; if <-10%, it is antagonistic effect.

[0077] 3. Results and analysis 3.1 Effect on the safety of silkworm growth During the whole feeding period of 5th instar, no poisoning phenomena such as refusal to eat, abnormal movement and death occurred in all treatment groups of silkworms, and the weight gain trend of silkworms in each group was basically consistent with that of the CK group, with no obvious difference (data not listed). It showed that the single use and combination of each additive were safe and non-toxic to the growth and development of silkworms, and no adverse effects or abnormal weight gain occurred.

[0078] 3.2 Effect of different treatments on cocoon layer amount of silkworm and synergistic effect analysis The effect of each treatment group on cocoon layer amount of golden cocoon variety is shown in Table 5.

[0079] The above experimental results showed that in the single agent treatment, the cocoon layer amount of CAN group was significantly higher than that of CK group (promotion rate 5.81%, P<0.05), while there was no significant difference between SP group and GABA group and CK group, indicating that the addition of silk sericin-derived small peptides or GABA alone had no significant improvement effect on cocoon layer amount.

[0080] In the binary combination group, the cocoon shell weight of the CAN+SP group was significantly higher than that of the CK group (P<0.05) and the CAN single agent group (P<0.01). According to the Bliss model, the theoretical expected promotion rate was 9.76%, and the measured value was 11.17% higher than the theoretical value, exceeding the 10% synergy threshold, indicating that canthaxanthin extract and silk sericin-derived small peptides had synergistic effects on promoting cocoon shell weight. The cocoon shell weight of the CAN+GABA group was also significantly higher than that of the CK group, but the theoretical expected promotion rate was 8.22%, and the difference between the measured value and the theoretical value was +0.62%, showing an additive effect. The SP+GABA group had no significant difference from the CK group, and the measured promotion rate of 5.35% was close to the theoretical value of 6.64%, also showing an additive effect.

[0081] For the ternary combination group (CAN+SP+GABA), the measured promotion rate of cocoon shell weight was 25.58%, significantly higher than that of the CK group (P<0.01) and the CAN single agent group (P<0.01), and also significantly higher than that of the CAN+SP binary synergistic group (P<0.05). Considering the combined effect of canthaxanthin extract (CAN) and the binary combination (SP+GABA), the theoretical expected promotion rate calculated based on the Bliss model was 10.85%, and the measured value was 14.73% higher than the theoretical value, exceeding the 10% synergy threshold, indicating that the ternary combination also had synergistic effects.

[0082] 3.3 Effects of different treatments on egg production of silkworm and analysis of synergistic effects The effects of different treatments on egg production of the golden cocoon variety are shown in Table 6.

[0083] From the determination results of egg production, the CAN group in the single agent treatment was significantly higher than the CK group (promotion rate 8.56%, P<0.05), while the SP group and the GABA group had no significant difference from the CK group.

[0084] In the binary combination group, the cocoon shell weight of the CAN+SP group was significantly higher than that of the CK group (P<0.05) and the CAN single agent group (P<0.01). Compared with the theoretical expected value of 12.60% calculated by the Bliss model, the measured value was 6.80% higher, and the difference did not exceed the 10% synergy threshold, showing an additive effect. The cocoon shell weight of the CAN+GABA group was also significantly higher than that of the CK group (promotion rate 12.00%, P<0.05), but the theoretical expected value was 13.70%, and the measured value was slightly lower, showing an additive effect. The SP+GABA group had no significant difference from the CK group, and the measured promotion rate of 7.49% was close to the theoretical value of 9.79%, also showing an additive effect.

[0085] For the ternary complex group (CAN+SP+GABA), the actual promotion rate of egg production was 22.65%, which was significantly higher than that of the CK group (P<0.01) and the CAN single dose group (P<0.01), and was also significantly higher than that of other binary complex groups (P<0.05). Considering the combined effect of canthaxanthin extract (CAN) and binary combination (SP+GABA), the theoretical expected promotion rate calculated based on the Bliss model was 15.41%, which was 7.24 percentage points higher than the actual value. The difference did not exceed the synergy threshold of 10%, and it showed an additive effect.

[0086] 3.4 Effect on cocoon color The color of the cocoon of each treatment group was observed, and the results were consistent with Example 1. The cocoon produced by all treatment groups containing canthaxanthin extract (the CAN single dose group and each complex group) was orange red, which was significantly different from the golden yellow of the CK group, and there was no significant difference in cocoon color among the complex groups, indicating that the addition of silk sericin-derived small peptides and GABA did not affect the coloring effect of canthaxanthin.

[0087] The following conclusions were drawn from the systematic comparative experiments and Bliss synergy effect analysis in this example: canthaxanthin extract and silk sericin-derived small peptides have a synergistic effect on improving cocoon layer weight; in terms of improving egg production, the binary combination shows an additive effect. The binary combination of canthaxanthin extract and GABA shows an additive effect in improving cocoon layer weight and egg production. On the basis of the synergistic effect of canthaxanthin extract and silk sericin-derived small peptides, further adding GABA (i.e. CAN+SP+GABA ternary complex) shows a synergistic effect in cocoon layer weight and an additive effect in egg production, but the ternary combination is significantly better than each binary complex group in both indicators. Therefore, the complex feed additive containing canthaxanthin extract, silk sericin-derived small peptides and γ-aminobutyric acid provides a technical solution for synergistically improving the economic traits of the silkworm.

[0088] The above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, which should be covered by the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are well-known technologies.

Claims

1. The application of canthaxanthin extract in silkworm farming, characterized in that, The application involves feeding silkworms with canthaxanthin extract to increase their egg production and / or produce orange-red cocoons.

2. A feed additive for silkworm rearing, characterized in that, The feed additive includes canthaxanthin extract.

3. The feed additive according to claim 2, characterized in that, The feed additive also includes any one or a mixture of two of the following: sericin-derived small peptides and / or γ-aminobutyric acid.

4. The feed additive according to claim 3, characterized in that, The sericin-derived small peptides are prepared by a method comprising the following steps: (a) The sericin or silk processing by-products rich in sericin are subjected to preliminary alkali treatment under the following conditions: pH 9.0-11.0, temperature 50℃-70℃, time 0.5-2 hours; (b) Adjust the product of step (a) to neutral, add a protease for enzymatic hydrolysis, wherein the protease is an alkaline protease or a complex protease, the hydrolysis temperature is 45℃-55℃, and the time is 3-6 hours. (c) The enzymatic hydrolysate is inactivated, filtered, concentrated and dried to obtain small peptides derived from sericin with a molecular weight of less than 3000 Da.

5. The feed additive according to claim 3 or 4, characterized in that, The weight ratio of the canthaxanthin extract, sericin-derived small peptides, and γ-aminobutyric acid is 1:(0.03~1):(0.03~1).

6. The feed additive according to any one of claims 2 to 5, characterized in that, Its dosage form can be either a solid dosage form or a liquid dosage form.

7. The use of the feed additive according to any one of claims 2 to 6 in the preparation of feed for silkworms.

8. The application according to claim 7, characterized in that, The application concentration of the additive in the feed is: 200-300 mg per 100g of feed, calculated as canthaxanthin extract.

9. The application according to claim 7, characterized in that, The feeding stage for supplemental feeding is the entire 5th instar of silkworms or the middle and late stages of the 5th instar.

10. The use of the feed additive according to any one of claims 2 to 6 in improving the economic traits of silkworms or in the production of orange-red silkworm cocoons, wherein, The economic traits mentioned are one or more of the following: increased egg production, increased cocoon layer weight, increased total cocoon weight, and increased pupa weight.

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