Bombyx mori breeding method for obtaining low-fat low-cholesterol high-quality silkworm chrysalis

Abstract

The invention provides a silkworm breeding method for obtaining low-fat low-cholesterol high-quality silkworm chrysalis, and belongs to the technical field of silkworm chrysalis production. According to the method, an acetate solution with the concentration of 5-15 mM is sprayed to the surfaces of mulberry leaves and naturally aired, the spraying amount is 0.06-0.2 mL / g of mulberry leaves, after silkworms are raised to the age of 5 and grow, treated mulberry leaves are fed in the morning and normal mulberry leaves are fed in the afternoon every day, and pupae are taken after matured silkworms cocoon and pupate for 4-7 days. According to the method, the crude fat content of the silkworm chrysalis can be reduced by 17.14%, the crude protein content can be increased by 15.2%, the triglyceride content and the cholesterol content can be reduced by 20.32% and 66.53% respectively, the nutritional quality of the silkworm chrysalis is remarkably optimized, and a high-quality silkworm chrysalis raw material is provided for the fields of eating, feeding and pharmacy.

Description

Technical Field

[0001] This invention belongs to the field of silkworm pupa production technology, and in particular relates to a method for raising silkworms to obtain high-quality silkworm pupae with low fat and low cholesterol. Background Technology

[0002] Silkworm breeding, a distinctive industry with a history spanning thousands of years in my country, occupies an important position in the silk textile industry. Silkworm pupae, a major byproduct of the silk reeling industry, possess significant resource advantages: their protein content is over 50%, containing all 18 essential amino acids, and their amino acid composition is highly similar to that of high-quality proteins such as eggs and milk. They also have a short breeding cycle (4-6 generations can be raised annually) and high bioconversion efficiency (approximately 0.15 kg of silkworm pupae can be produced from every kilogram of mulberry leaves), making them a highly promising insect protein source.

[0003] However, the industrial application of silkworm pupae currently faces significant bottlenecks: the fat content of natural silkworm pupae is typically between 35% and 40%, and the cholesterol content is as high as 0.9-1.2 mmol / 100g, far exceeding that of common high-cholesterol foods such as egg yolks and pig brains. This nutritional characteristic not only limits its application in the high-end food sector (such as healthy snacks and functional foods), but also leads to the risk of elevated blood lipids in animals after consumption during feed processing.

[0004] In recent years, with the increasing global shortage of protein resources and the surge in consumer demand for low-fat, low-cholesterol foods, improving the nutritional quality of silkworm pupae has become crucial for industry upgrading. Existing technologies, such as genetically selecting and breeding silkworm varieties, are time-consuming, costly, and may affect silk production; while physical or chemical degreasing can reduce fat content, it damages protein structure and introduces safety hazards. Therefore, there is an urgent need to develop a green and efficient nutritional regulation method to directly optimize the nutrient composition of silkworm pupae during the rearing process. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for raising silkworms to obtain high-quality silkworm pupae with low fat and low cholesterol, thereby optimizing the nutritional quality of edible silkworm pupae.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of acetate in the production of high-protein, low-fat, and low-cholesterol silkworm pupae during silkworm rearing.

[0008] The present invention also provides a method for preparing mulberry leaves for silkworm rearing, comprising the following steps: spraying an acetate solution onto the surface of the mulberry leaves and air-drying them naturally.

[0009] Preferably, the acetate solution is a sodium acetate solution with a concentration of 5-15 mM and a spraying rate of 0.06-0.2 mL / g mulberry leaves.

[0010] The present invention correspondingly provides mulberry leaves for silkworm rearing prepared by the above preparation method.

[0011] The present invention also provides a method for raising silkworms, comprising the following steps: after the silkworms are raised normally until they reach the 5th instar, they are fed the above-mentioned mulberry leaves until the mature silkworms no longer eat mulberry leaves.

[0012] Preferably, the above-mentioned mulberry leaves are fed in the morning, and normal mulberry leaves are fed in the afternoon.

[0013] This invention also provides a method for preparing high-protein, low-fat, and low-cholesterol silkworm pupae. The silkworms are raised according to the above-mentioned feeding method, and after 4-7 days of mature silkworms, cocooning, and pupation, the cocoons are peeled off and the pupae are harvested.

[0014] Preferably, after the silkworm pupae are removed from the cocoons, the pupae undergo further processing, including fresh packaging, frozen preservation, freeze drying, hot air drying, salt preservation, or canning.

[0015] The present invention provides high-protein, low-fat, and low-cholesterol silkworm pupae prepared by the above-described method.

[0016] The present invention also provides the application of the above-mentioned high-protein, low-fat, low-cholesterol silkworm pupae in food, feed, or pharmaceuticals.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention, by feeding silkworms with acetate, significantly reduces the fat, cholesterol, and triglyceride content of silkworm pupae while increasing protein content, creating an ideal "high-protein, low-fat" nutritional structure that meets the nutritional requirements of modern health foods. The regulatory effect of acetate far surpasses that of sodium propionate and probiotics, effectively addressing the risk of consuming high-cholesterol silkworm pupae.

[0019] This invention utilizes a foliar spraying method with sodium acetate solution, requiring no genetic modification or chemical extraction. It is simple to operate, low-cost, and suitable for large-scale implementation. Furthermore, it does not affect the normal growth cycle of silkworms, ensuring that key production indicators such as cocoon layer rate and pupation rate remain unaffected, thus guaranteeing that the main silk reeling business is not impacted.

[0020] The high-protein, low-fat, and low-cholesterol silkworm pupae prepared by this invention can be directly used as a high-end food ingredient to meet the protein needs of special groups such as fitness enthusiasts and patients with hyperlipidemia. The silkworm pupae powder, produced through freeze-drying, can be used as a functional additive in baked goods, nutritional supplements, and other fields. In the feed industry, low-fat silkworm pupae protein powder can replace protein raw materials in livestock, poultry, and aquaculture, reducing costs and avoiding the risk of elevated blood lipids in animals. Its extracts can also be used to develop health foods that regulate human blood lipids. Attached Figure Description

[0021] Figure 1 Changes in relevant indicators of antioxidant activity in the hemolymph of silkworm larvae after supplementation with acetate;

[0022] Figure 2 The changes in the activity of intestinal digestive enzymes in silkworm larvae after feeding them acetate;

[0023] Figure 3 To investigate the differences in gut microbiota composition in silkworm larvae after supplementation with acetate;

[0024] Figure 4 Analysis of differential gene expression patterns in the gut of silkworm larvae 5 days after supplementation with acetate;

[0025] Figure 5 Annotation analysis of differentially expressed genes in the gut of silkworm larvae on day 5 after supplemental feeding with acetate;

[0026] Figure 6 The crude fat and crude protein content of silkworm pupae after adding acetate;

[0027] Figure 7 The cholesterol and triglyceride content of silkworm pupae after adding acetate;

[0028] Figure 8 The effects of different feeding treatments on the crude protein and crude fat content of silkworm pupae;

[0029] Figure 9 The effect of different feeding treatments on the total cholesterol content of silkworm pupae;

[0030] Figure 10 The effects of different feeding treatments on the triglyceride content of silkworm pupae. Detailed Implementation

[0031] This invention provides the application of acetate in the production of high-protein, low-fat, and low-cholesterol silkworm pupae during silkworm rearing.

[0032] The present invention also provides a method for preparing mulberry leaves for silkworm rearing, comprising the following steps: spraying an acetate solution onto the surface of the mulberry leaves and air-drying them naturally; preferably, drying them at room temperature for 1 hour.

[0033] In this invention, the preferred acetate solution is a sodium acetate solution with a concentration of 5-15 mM, more preferably 6, 7, 8, 9, 10, 11, 12, 13, or 14 mM, and the spraying amount is 0.06-0.2 mL / g mulberry leaves, more preferably 0.08, 0.1, 0.12, 0.14, 0.16, or 0.18 mL / g. As one possible implementation, a 10 mM sodium acetate solution is prepared in advance, and before feeding, 50 mL of the sodium acetate solution is sprayed evenly over every 500 g of mulberry leaves.

[0034] The present invention provides mulberry leaves for silkworm rearing prepared by the above preparation method, which are used directly for silkworm rearing.

[0035] The present invention also provides a method for raising silkworms, comprising the following steps: after the silkworms are raised normally until they reach the 5th instar, they are fed the above-mentioned mulberry leaves until the mature silkworms no longer eat mulberry leaves.

[0036] In this invention, it is preferable to feed the above-mentioned mulberry leaves in the morning and feed normal mulberry leaves in the afternoon.

[0037] This invention also provides a method for preparing high-protein, low-fat, and low-cholesterol silkworm pupae. The silkworms are fed according to the above-mentioned feeding method. After the silkworms mature, spin cocoons, and pupate for 4-7 days, the cocoons are peeled off and the pupae are taken out. Preferably, the pupae are peeled off and taken out 4 or 5 days after pupation.

[0038] In this invention, preferably after removing the cocoons and pupae, the silkworm pupae undergo further processing, including fresh packaging, freezing, freeze-drying, hot air drying, salting, or canning. More preferably, appropriate processing methods are selected based on requirements. As optional implementation methods, fresh packaging is used for short-distance transportation; freezing is used for long-distance sales or as processing raw materials; freeze-drying is used for producing high-end snacks and health products; and drying is used for producing low-end snacks and feed. This invention does not specifically limit the specific processes and parameters of the above-mentioned processing; methods commonly used in the art can be employed.

[0039] The present invention provides high-protein, low-fat, and low-cholesterol silkworm pupae prepared by the above-described method.

[0040] The present invention also provides the application of the above-mentioned high-protein, low-fat, low-cholesterol silkworm pupae in food, feed or pharmaceuticals; preferably including whole silkworm pupae, silkworm pupa powder or silkworm pupa extract.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] A method for preparing mulberry leaves for silkworm rearing is as follows:

[0044] Prepare a 10mM sodium acetate solution for later use; before feeding silkworms, spray the sodium acetate solution onto the surface of mulberry leaves at a rate of 0.1mL / g and allow them to air dry naturally.

[0045] Example 2

[0046] A method for preparing mulberry leaves for silkworm rearing is as follows:

[0047] Prepare a 5mM sodium acetate solution for later use; before feeding silkworms, spray the sodium acetate solution onto the surface of mulberry leaves at a rate of 0.2mL / g and allow them to air dry naturally.

[0048] Example 3

[0049] A method for preparing mulberry leaves for silkworm rearing is as follows:

[0050] Prepare a 15mM sodium acetate solution for later use; before feeding silkworms, spray the sodium acetate solution onto the surface of mulberry leaves at a rate of 0.06mL / g and allow them to air dry naturally.

[0051] Example 4

[0052] A high-protein, low-fat, low-cholesterol silkworm pupa is prepared as follows:

[0053] After the silkworms are raised normally to the 5th instar, they are fed mulberry leaves as described in Example 1 every morning and normal mulberry leaves in the afternoon until the mature silkworms stop eating mulberry leaves. Four days after the mature silkworms spin cocoons and pupate, the cocoons are peeled off and the pupae are taken out.

[0054] Experimental Example 1

[0055] Silkworms (ZOX variety) were fed mulberry leaves (AC) as described in Example 4, supplemented with the same mulberry leaves (AC) as in Example 1. A control group (CON) was used, sprayed with distilled water. Each group consisted of three sections, with 150 silkworm larvae per section. The larvae were tested for various indicators on days 1, 3, and 5 after supplementation. These included:

[0056] (1) The activity of T-SOD and T-AOC in the hemolymph of silkworm larvae was detected. The assay method was performed in accordance with the instructions of the T-SOD and T-AOC detection kit of Nanjing Jiancheng Biotechnology Co., Ltd.

[0057] (2) The activities of trypsin, lipase and amylase in the intestine of silkworm larvae were detected. The assay method was performed in accordance with the instructions of the trypsin, lipase and α-amylase assay kit of Nanjing Jiancheng Biotechnology Co., Ltd.

[0058] (3) Samples of the midgut of silkworm larvae were subjected to 16s high-throughput sequencing. The sequencing results were clustered at a similarity level of 97.0% using Usearch software to obtain the number of operational taxonomic units (OTUs) for different samples. The Naive Bayes classifier was used to annotate the feature sequences using Silva.138 as a reference database. The bacterial community composition of each sample was statistically analyzed at different classification levels to draw a bacterial community structure diagram at the genus level of the sample.

[0059] (4) On day 5, midgut tissue of silkworm larvae was collected and RNA-seq high-throughput sequencing was performed using the Illumina high-throughput sequencing platform. The sequencing data of each sample were compared with the silkworm reference genome sequence (Bombyx_mori.v1.0.genome.fa) for sequence alignment and expression quantification, and differentially expressed genes were screened.

[0060] Experimental results:

[0061] (1) As Figure 1 As shown, starting from the 3rd day of feeding with mulberry leaves containing acetate, the activities of T-SOD and T-AOC in the hemolymph of silkworm larvae were significantly increased (p<0.05), indicating that adding acetate can significantly improve the antioxidant capacity of silkworm larvae.

[0062] (2) Figure 2 As shown, the activities of trypsin and lipase in the intestinal digestive fluid of silkworm larvae in the acetate-fed group were significantly higher than those in the control group (p < 0.05), while the activity of α-amylase did not change significantly (p > 0.05). This indicates that acetate feeding significantly improved the digestibility of protein and fat in the intestine of silkworm larvae, but had no significant effect on starch digestion.

[0063] (3) Figure 3 As shown, the control group and the acetate-added group had a total of 684 OTUs. In the control group (CON1D, CON3D, CON5D) and the acetate group (AC1D, AC3D, AC5D) on days 1-5, 1801, 724, 387, 870, 735, and 560 OTUs respectively were unique to each group. The data showed that the total number of OTUs decreased with increasing age (five years), but the total number of OTUs was higher on days 3 and 5 after acetate addition than in the control group. Figure 3 (Left). In terms of species composition, the relative abundance of *Staphylococcus*, *Oligotrophomonas*, and *Rhizobium* increased after the addition of acetate compared to the control group at the same time point. Figure 3 (Right). These results indicate that with the increase in acetate feeding time, the number of species in the intestinal flora of silkworm larvae increased significantly compared with the control at the same time point, and the composition of the flora also changed significantly.

[0064] (4) Figure 4 and Figure 5As shown, on day 5 of feeding, 45 differentially expressed genes were found between the acetate group and the control group, including 34 upregulated genes and 11 downregulated genes. Enrichment of expression patterns and KEGG analysis of these differentially expressed genes revealed that they were mainly enriched in multiple pathways such as pentose and glucuronic acid metabolism, vitamin B6 metabolism, drug metabolism, lysosomes, insect hormones, and amino acid synthesis and metabolism. This indicates that feeding with acetate significantly affected the expression activities of genes related to multiple pathways such as glycolysis, detoxification, larval growth, and nutrition.

[0065] Experimental Example 2

[0066] Silkworms (ZOX variety) were reared according to Example 4, with distilled water spraying serving as the control group. Each group consisted of three sections, with 150 silkworm larvae per section. The larvae were reared until they reached maturity and spun cocoons. The pupae, which had been pupated for 4 days, were freeze-dried and their basic nutrient composition was determined. The determination methods were as follows: crude protein content was determined by the Kjeldahl method according to GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food"; fat content was determined by the Soxhlet extraction method according to GB5009.6-2016 "National Food Safety Standard - Determination of Fat in Food"; total cholesterol (TCH / T-CHO(A111-1-1)) and triglycerides (TG(A110-1-1)) were determined using the methods specified in the Nanjing Jiancheng reagent kit.

[0067] Experimental results:

[0068] like Figure 6 As shown, after adding acetate, the crude fat content of silkworm pupae decreased from 38.8% to 32.15%, which was significantly lower than the control group by 17.14% (P<0.05), while the crude protein content of silkworm pupae was 51.96%, which was significantly higher than the control group by 15.2% (P<0.05).

[0069] like Figure 7 As shown, compared with the control group, the triglyceride and total cholesterol content in silkworm pupae after adding acetate were 3.422 mmol / L and 0.328 mmol / L, respectively, which decreased by 20.32% and 66.53% compared with the control group (P<0.05).

[0070] Experimental Example 3

[0071] Silkworms (ZOX variety) were raised according to Example 4. A group sprayed with the same amount of sodium propionate and a probiotic group (Lactobacillus casei) were also included, with distilled water serving as a control group. Each group consisted of three sections, with 150 silkworm larvae per section. The larvae were raised until they reached maturity and spun cocoons. The pupae, after four days of pupation, were freeze-dried, and their basic nutrient composition was determined. Same as in Example 2.

[0072] Experimental results:

[0073] like Figure 8 As shown, the addition of acetate, sodium propionate, and probiotics significantly increased the protein content of silkworm pupae (P < 0.05), with the acetate group showing the best effect, increasing crude protein by 15.21%. The crude fat content of silkworm pupae was significantly reduced (P < 0.05), with the acetate group showing the most significant reduction, decreasing crude fat content by 17.14% compared to the control group.

[0074] like Figure 9 As shown, the total cholesterol content of the three feeding groups was significantly lower than that of the control group. The cholesterol content of the acetate feeding group was also significantly lower than that of the other two feeding groups (P<0.05). Compared with the control, the total cholesterol content of silkworm pupae in the acetate feeding group decreased by 66% (P<0.05).

[0075] like Figure 10 As shown, the triglyceride content in silkworm pupae in the three feeding groups was significantly lower than that in the control group. Compared with the control, the triglyceride content in silkworm pupae in the acetate, propionate, and probiotic feeding groups decreased by 20.32%, 17.59%, and 22.45%, respectively (P < 0.05).

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of acetate in the production of high-protein, low-fat, and low-cholesterol silkworm pupae.

2. A method for preparing mulberry leaves for silkworm rearing, characterized in that, Includes the following steps: Spray the surface of mulberry leaves with an acetate solution and let them air dry naturally.

3. The preparation method according to claim 2, characterized in that, The acetate solution is a sodium acetate solution with a concentration of 5-15 mM and a spraying rate of 0.06-0.2 mL / g mulberry leaves.

4. Mulberry leaves for silkworm rearing prepared by the method described in claim 2 or 3.

5. A method for raising silkworms, characterized in that, Includes the following steps: After the silkworms are normally raised to the 5th instar, they are fed the mulberry leaves as described in claim 4 until the mature silkworms stop eating the mulberry leaves.

6. The feeding method according to claim 5, characterized in that, Feed the animals with the specified mulberry leaves every morning and with normal mulberry leaves every afternoon.

7. A method for preparing high-protein, low-fat, and low-cholesterol silkworm pupae, characterized in that, Silkworms are raised according to the feeding method described in claim 5 or 6. After 4-7 days from maturity, cocoon formation, and pupation, the cocoons are peeled off and the pupae are taken.

8. The preparation method according to claim 7, characterized in that, After the silkworm pupae are removed from the cocoons, they undergo further processing, including fresh packaging, frozen preservation, freeze drying, hot air drying, salt preservation, or canning.

9. The high-protein, low-fat, low-cholesterol silkworm pupae prepared by the preparation method according to claim 7 or 8.

10. The use of the high-protein, low-fat, low-cholesterol silkworm pupae as described in claim 9 in food, feed, or pharmaceutical applications.

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