Method for improving growth and nutritional traits of silkworm and application thereof

Abstract

The application provides a method for improving the growth and nutritional traits of domestic silkworms and application thereof, and relates to the technical field of domestic silkworm breeding. The application adds Lactobacillus rhamnosus to the domestic silkworms when the domestic silkworms are raised to the fourth instar, and the feeding dose of the Lactobacillus rhamnosus is 0.05-0.1 ml per silkworm; and / or the application adds Lactobacillus rhamnosus to the domestic silkworms when the domestic silkworms are raised to the fifth instar, and the feeding dose of the Lactobacillus rhamnosus is 0.1-0.2 ml per silkworm; the viable bacterial count of the Lactobacillus rhamnosus in each milliliter is 10 6 -10 8 CFU / mL. The application adds a certain dose of Lactobacillus rhamnosus to the domestic silkworms during the feeding process, so as to improve the viability of the domestic silkworms and the nutritional composition of the silkworm pupae, and the method has good application potential in the disease prevention of domestic silkworms and the screening and preparation of high-quality edible silkworm pupae.

Description

Technical Field

[0001] This invention belongs to the field of silkworm breeding technology, specifically a method for improving the growth and nutritional traits of silkworms and its application. Background Technology

[0002] Silkworm farming is a traditional and distinctive industry in my country. In recent years, its breeding model has undergone tremendous changes, shifting from traditional small-scale, scattered farming to large-scale, labor-saving operations. Traditional mulberry leaf breeding is also gradually transitioning to artificial feed breeding. Furthermore, the development of smart agriculture has led to frequent cases of micro-agricultural poisoning from mulberry leaves caused by drone-based pest control. Current methods for silkworm disease prevention and control include breeding resistant varieties and chemical treatments. However, there is a lack of highly resistant materials, making it difficult to breed new, highly resistant, and high-quality silkworm varieties. Moreover, silkworm diseases are caused by complex pathogens, have short lifespans, and are difficult to treat with effective drugs. Therefore, disease prevention and control are challenging in large-scale silkworm farming. In addition, silkworm pupae, a byproduct of silk reeling, are a high-quality insect food due to their high protein content, balanced nutritional structure, short breeding cycle, and abundant resources. However, silkworm pupae have relatively high fat content and low levels of functional peptides and other functional active substances under normal circumstances. Further improvements to the nutritional composition of silkworm pupae to better meet people's health needs and align with current development demands under the broader food concept framework are needed.

[0003] In recent years, research on probiotics has attracted much attention. Studies have found that probiotics can improve the host's gut health, enhance the host's immunity, regulate the host's glucose and lipid metabolism, and optimize nutritional components. However, there are few reports on whether supplementing with probiotics will affect the growth, metabolism, and nutrient composition of silkworms and their pupae. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for improving the growth and nutritional traits of silkworms, which can effectively improve the vitality of silkworm larvae and improve the nutrient composition of silkworm pupae.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for improving the growth and nutritional traits of silkworms, comprising the following steps:

[0007] When silkworm larvae are raised to the fourth instar, they are fed with Lactobacillus rhamnosus. The dosage of Lactobacillus rhamnosus is 0.05-0.1 ml per fourth instar silkworm.

[0008] And / or, when silkworm larvae are raised to the fifth instar, they are fed with Lactobacillus rhamnosus, and the dosage of Lactobacillus rhamnosus is 0.1-0.2 ml / silkworm for the fifth instar.

[0009] The viable count of Lactobacillus rhamnosus per milliliter is 10. 6 -108 CFU / mL.

[0010] Preferably, the Latin name of the Lactobacillus rhamnosus is Lactobacillus rhamnosus, and the accession number is ATCC53103.

[0011] Preferably, the method of adding Lactobacillus rhamnosus is to spray Lactobacillus rhamnosus bacterial solution onto the surface of mulberry leaves.

[0012] Preferably, the method for preparing the Lactobacillus rhamnosus includes the following steps:

[0013] Lactobacillus rhamnosus was activated and cultured to obtain Lactobacillus rhamnosus bacterial solution;

[0014] The Lactobacillus rhamnosus bacterial suspension was diluted to obtain a diluted bacterial suspension;

[0015] Add mulberry oligosaccharides and Tween-80 to the diluted bacterial solution for later use.

[0016] More preferably, the culture medium for the Lactobacillus rhamnosus is MRS medium.

[0017] More preferably, the culture conditions for the Lactobacillus rhamnosus are 32-37°C, with shaking at 180 rpm overnight.

[0018] More preferably, the Lactobacillus rhamnosus bacterial solution is diluted to 10... 6 -10 8 CFU / mL.

[0019] More preferably, the amount of mulberry oligosaccharide added to the diluted Lactobacillus rhamnosus bacterial solution is 0.4-0.6 g / 100 mL; and the amount of Tween-80 added is 0.1-0.5 ml / 100 mL.

[0020] The present invention also provides an application of the method in cultivating high-quality edible silkworm pupae.

[0021] The present invention also provides an application of the method in the prevention and control of silkworm diseases.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) This invention provides a method for improving the growth and nutritional traits of silkworms by adding probiotics. This method involves adding a certain dose of Lactobacillus rhamnosus (accession number ATCC53103) to the silkworms during the rearing process, which can effectively improve the vitality of silkworm larvae and improve the nutrient composition of silkworm pupae.

[0024] (2) Compared with the group not fed probiotics, the T-SOD activity in the hemolymph of 5th instar silkworms fed with probiotics was significantly higher than that of the control group except on days 2 and 6 (P < 0.05); the T-AOC capacity was higher or significantly higher than that of the control group except on day 5 (P < 0.05); and the DPPH free radical scavenging capacity was higher or significantly higher than that of the control group from day 1 to day 6 (P < 0.05). Except on day 5 or 6, the lipase and trypsin activities in the intestine of 5th instar silkworms fed with probiotics were significantly higher than those of the control group (P < 0.05). On days 4 and 6 after feeding, the relative expression levels of antimicrobial peptides BmAttacin1, BmDefensinA, and BmLebocin3 in the fat body of silkworm larvae were significantly higher than those in the control group (P < 0.05).

[0025] (3) The vitality of larvae was significantly improved after feeding with Lactobacillus rhamnosus. The cocooning rate of mature silkworm larvae reached 78.59%, which was significantly higher than that of the Bifidobacterium breve group and the control group (P<0.05). The crude protein content of the Lactobacillus rhamnosus group was significantly higher than that of the control group and the Bifidobacterium breve group (P<0.05), while the crude fat content was significantly lower than that of the Bifidobacterium breve group (P<0.05), and it also showed a decreasing trend compared with the control group. The saturated fatty acid content in silkworm pupae fed with Lactobacillus rhamnosus was significantly reduced, while the content of unsaturated fatty acids, ω-3 and essential fatty acids was significantly higher than that of the control group and the Bifidobacterium breve group (P<0.05). Attached Figure Description

[0026] Figure 1 The T-SOD activity of hemolymph in silkworm larvae after feeding with Lactobacillus rhamnosus was shown in the figure. CK and LR represent the control group and Lactobacillus rhamnosus group, respectively. * indicates a significant difference at the 0.05 level.

[0027] Figure 2 The T-AOC capacity of silkworm larvae hemolymph after being fed Lactobacillus rhamnosus was shown in the figure. CK and LR represent the control group and Lactobacillus rhamnosus group, respectively. * indicates a significant difference at the 0.05 level.

[0028] Figure 3 To illustrate the DPPH free radical scavenging capacity of silkworm larvae hemolymph after feeding with Lactobacillus rhamnosus, the figure shows that CK and LR represent the control group and the Lactobacillus rhamnosus group, respectively, with * indicating a significant difference at the 0.05 level.

[0029] Figure 4 The figure shows the changes in trypsin activity in the intestinal fluid of silkworms after feeding with Lactobacillus rhamnosus. CK and LR in the figure represent the control group and the Lactobacillus rhamnosus group, respectively, and different lowercase letters indicate significant differences at the 0.05 level.

[0030] Figure 5The figure shows the changes in lipase activity in the intestinal fluid of silkworms after feeding with Lactobacillus rhamnosus. CK and LR represent the control group and the Lactobacillus rhamnosus group, respectively. Different lowercase letters indicate significant differences at the 0.05 level, and different uppercase letters indicate significant differences at the 0.01 level.

[0031] Figure 6 The figure shows the relative expression level of the antimicrobial peptide gene BmAttacin1 in the fat body of silkworm after feeding with Lactobacillus rhamnosus. CK and LR in the figure represent the control group and the Lactobacillus rhamnosus group, respectively, and different lowercase letters indicate significant differences at the 0.05 level.

[0032] Figure 7 The change in the relative mRNA expression level of the antimicrobial peptide gene BmDefensinA in the fat body of silkworm after feeding with Lactobacillus rhamnosus was shown in the figure. CK and LR represent the control group and the Lactobacillus rhamnosus group, respectively. Different lowercase letters indicate significant differences at the 0.05 level.

[0033] Figure 8 The figure shows the relative expression level of the antimicrobial peptide gene BmLebocin3 in the fat body of silkworm after feeding with Lactobacillus rhamnosus. In the figure, CK and LR represent the control group and the Lactobacillus rhamnosus group, respectively, and different lowercase letters represent significant differences at the 0.05 level.

[0034] Figure 9 The effects of different probiotic supplementation on the viability of silkworm larvae; in the figure, CK, LR and Bb represent the control group, Lactobacillus rhamnosus group and Bifidobacterium breve group, respectively, and different lowercase letters indicate significant differences at the 0.05 level;

[0035] Figure 10 The effects of adding different probiotics to the diet on the fatty acid composition of silkworm pupae were shown in the figure. CK, LR and Bb represent the control group, Lactobacillus rhamnosus group and Bifidobacterium breve group, respectively. Lowercase letters indicate significant differences among the different feeding groups (p < 0.05). Detailed Implementation

[0036] This invention provides a method for improving the growth and nutritional traits of silkworms, comprising the following steps:

[0037] When silkworm larvae are raised to the fourth instar, they are fed with Lactobacillus rhamnosus. The preferred dosage of Lactobacillus rhamnosus is 0.05-0.1 ml per fourth instar silkworm; the more preferred dosage is 0.1 ml per fourth instar silkworm.

[0038] And / or, when silkworm larvae are raised to the fifth instar, they are fed with Lactobacillus rhamnosus, and the dosage of Lactobacillus rhamnosus is 0.1-0.2 ml / silkworm for the fifth instar; more preferably, the dosage of Lactobacillus rhamnosus is 0.15 ml / silkworm for the fifth instar.

[0039] The optimal viable count of Lactobacillus rhamnosus per milliliter is 10. 6 -10 8 CFU / mL; more preferably 10 7 CFU / mL.

[0040] In this invention, the Latin name of the *Lactobacillus rhamnosus* is *Lactobacillus rhamnosus*, with accession number ATCC53103. This invention does not impose any special restrictions on the source of the *Lactobacillus rhamnosus*, which can be obtained through conventional purchasing channels.

[0041] In this invention, there is no specific limitation on the timing of adding Lactobacillus rhamnosus. In a specific embodiment of this invention, it is preferable to add Lactobacillus rhamnosus once every morning. In this invention, the preferred method for adding Lactobacillus rhamnosus is to spray the Lactobacillus rhamnosus bacterial solution onto the surface of mulberry leaves.

[0042] In this invention, the method for preparing Lactobacillus rhamnosus preferably includes the following steps:

[0043] Lactobacillus rhamnosus was activated and cultured to obtain Lactobacillus rhamnosus bacterial solution;

[0044] The Lactobacillus rhamnosus bacterial suspension was diluted to obtain a diluted bacterial suspension;

[0045] Add mulberry oligosaccharides and Tween-80 to the diluted bacterial solution for later use.

[0046] In this invention, the activation method of *Lactobacillus rhamnosus* is not specifically limited, and activation can be performed according to the normal bacterial activation method. In this invention, as one feasible method, the activation method is preferably to remove the strain from -80°C, streak it on an MRS solid culture plate, incubate it upside down at 37°C for 48 hours, then pick a single colony, inoculate it onto 5 ml MRS liquid medium, and incubate it overnight at 37°C and 180 rpm.

[0047] In this invention, the culture medium for *Lactobacillus rhamnosus* is preferably MRS medium. The source of the MRS medium is not specifically limited in this invention and can be purchased from commercially available products. In a specific embodiment of this invention, the MRS medium was purchased from Guangdong Huankai Biotechnology Co., Ltd., product number 027312.

[0048] In this invention, the *Lactobacillus rhamnosus* is inoculated into MRS medium for culture. In this invention, the inoculation concentration of *Lactobacillus rhamnosus* is preferably 0.5%-2%; more preferably 1%. In this invention, the "%" in the inoculation concentration of *Lactobacillus rhamnosus* of 0.5%-2% refers to a volume percentage.

[0049] In this invention, the preferred culture conditions for Lactobacillus rhamnosus are 32-37°C and overnight culture with shaking at 180 rpm; more preferably, the culture conditions for Lactobacillus rhamnosus are 35°C and overnight culture with shaking at 180 rpm.

[0050] In this invention, the Lactobacillus rhamnosus bacterial solution is preferably diluted to 10. 6 -10 8 CFU / mL; more preferably diluted to 10 7 CFU / mL. In this invention, the diluent is not specifically limited and can be conventionally selected according to actual needs. In a specific embodiment of this invention, sterile water is preferably used as the diluent.

[0051] In this invention, the preferred amount of mulberry oligosaccharide added to the diluted Lactobacillus rhamnosus bacterial solution is 0.4-0.6 g / 100 mL; the preferred amount of Tween-80 added is 0.1-0.5 ml / 100 mL. More preferably, the amount of mulberry oligosaccharide added is 0.5 g / 100 mL; the amount of Tween-80 added is 0.3 ml / 100 mL. In this invention, the source of the mulberry oligosaccharide and Tween-80 is not specifically limited, and they can be purchased from conventional commercially available products.

[0052] This invention also provides an application of the method in cultivating high-quality edible silkworm pupae. The method of this invention can increase the crude protein content, as well as the content of unsaturated fatty acids, ω-3 fatty acids, and essential fatty acids in silkworm pupae, while reducing the content of saturated fatty acids and total sugars, thus improving the nutrient composition of silkworm pupae and having potential applications in cultivating high-quality edible silkworm pupae.

[0053] The present invention also provides an application of the method in the prevention and control of silkworm diseases.

[0054] 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.

[0055] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0056] Example 1

[0057] A method for improving the growth and nutritional traits of silkworms includes the following steps:

[0058] Activation and preparation of *Lactobacillus rhamnosus*: The strain stored at -80℃ was activated according to normal bacterial activation procedures, then inoculated at a 1% concentration into MRS medium and cultured overnight at 37℃ with shaking at 180 rpm. After counting with a hemocytometer, the bacterial suspension was diluted to 10⁻¹⁰ with sterile water. 7 CFU / mL, the amount of mulberry oligosaccharide added to the diluted bacterial solution is 0.5g / 100mL, and the amount of Tween-80 added is 0.1ml / 100mL, for later use.

[0059] When silkworm larvae reach the fourth instar, they should be fed with Lactobacillus rhamnosus once a day in the morning. The feeding method is as follows: spray the prepared Lactobacillus rhamnosus bacterial solution onto the surface of mulberry leaves, with a dosage of 0.1 ml per silkworm in the fourth instar.

[0060] Example 2

[0061] Unlike Example 1, when silkworm larvae are raised to the fifth instar, Lactobacillus rhamnosus is added to their diet, with a dosage of 0.15 ml per silkworm in the fifth instar.

[0062] Example 3

[0063] Unlike Example 1, the dosage for fourth-instar silkworms was 0.05 ml per silkworm.

[0064] Example 4

[0065] Unlike Example 1, the dosage for fourth-instar silkworms was 0.08 ml per silkworm.

[0066] Example 5

[0067] Unlike Example 2, the dosage for fifth-instar silkworms was 0.1 ml per silkworm.

[0068] Example 6

[0069] Unlike Example 2, the dosage for fifth-instar silkworms was 0.2 ml per silkworm.

[0070] Comparative Example 1

[0071] Unlike Example 1, this animal was not fed Lactobacillus rhamnosus and was fed normal mulberry leaves.

[0072] Comparative Example 2

[0073] Unlike Example 2, this animal was not fed Lactobacillus rhamnosus and was fed normal mulberry leaves.

[0074] Comparative Example 3

[0075] Unlike Example 1, 0.1 ml of Bifidobacterium breve was added to the diet of each silkworm. The Bifidobacterium breve was obtained by culturing on RCM medium, which was purchased from Qingdao Haibo Biotechnology Co., Ltd., product number HB0316.

[0076] Comparative Example 4

[0077] Unlike Example 2, 0.15 ml of Bifidobacterium breve was added to the diet of each silkworm. The Bifidobacterium breve was obtained by culturing on RCM medium, which was purchased from Qingdao Haibo Biotechnology Co., Ltd., product number HB0316.

[0078] Example 7

[0079] Hemolymph from silkworm larvae of Example 2 (fed with Lactobacillus rhamnosus) and Comparative Example 2 (without Lactobacillus rhamnosus) was collected. The T-SOD activity, T-AOC capacity, and DPPH free radical scavenging capacity of the hemolymph from larvae aged 1-6 days of the 5th instar were detected according to the instructions of the T-SOD, T-AOC, and DPPH detection kits from Nanjing Jiancheng Bioengineering Institute.

[0080] The results are as follows Figure 1-3 As shown: Compared with the group not fed with Lactobacillus rhamnosus (Ck), the group fed with Lactobacillus rhamnosus (LR) had significantly higher T-SOD activity in the hemolymph of 5-year-old infants, except on days 2 and 6 (P < 0.05). Figure 1 T-AOC capacity was higher or significantly higher than the control group except on day 5 (P < 0.05). Figure 2 The DPPH free radical scavenging capacity was higher than or significantly higher than the control group within 1-6 days (P<0.05). Figure 3 This indicates that supplementing the diet with Lactobacillus rhamnosus can increase the antioxidant index of hemolymph in silkworm larvae.

[0081] Example 8

[0082] Take the midgut fluid of silkworm larvae from Example 2 (fed with Lactobacillus rhamnosus) and Comparative Example 2 (without Lactobacillus rhamnosus), and test the trypsin and lipase activities in the inguinal fluid of larvae aged 1-6 days of the 5th instar according to the instructions of the trypsin and lipase detection kit from Nanjing Jiancheng Bioengineering Institute.

[0083] The results are as follows Figure 4-5 As shown: Compared with the group not fed Lactobacillus rhamnosus (Ck), except on day 5 or 6, the trypsin activity in the intestine of 5th instar silkworms fed Lactobacillus rhamnosus (LR) was significantly lower. Figure 4 ) and lipase activity ( Figure 5 The levels of both were significantly higher than those in the control group (P < 0.05). This indicates that supplementing the diet with Lactobacillus rhamnosus can improve the activity of intestinal digestive enzymes in silkworm larvae.

[0084] Example 9

[0085] Total RNA was extracted from the fat bodies of silkworm larvae from Example 2 (fed with Lactobacillus rhamnosus) and Comparative Example 2 (without Lactobacillus rhamnosus). The relative mRNA expression levels of the antimicrobial peptide genes BmAttacin1, BmDefensinA, and BmLebocin3 at 5 instars 2, 4, and 6 days were detected and analyzed using the TAKARA reverse transcription and quantitative fluorescence detection kit.

[0086] The results are as follows Figure 6-8 As shown: Compared with the control group (Ck) without Lactobacillus rhamnosus, on days 4 and 6 after supplementation with Lactobacillus rhamnosus, the antimicrobial peptide BmAttacin1 in the fat body of silkworm larvae was significantly higher. Figure 6 ), BmDefensinA ( Figure 7 ) and BmLebocin3 ( Figure 8 The relative expression level of mRNA in the control group was significantly higher than that in the control group (P < 0.05). This indicates that feeding with Lactobacillus rhamnosus can enhance the transcriptional activity of antimicrobial peptides in the fat body of silkworm larvae.

[0087] Example 10

[0088] Following the method described in Example 2, silkworm larvae were fed *Lactobacillus rhamnosus*, and following the method described in Comparative Example 4, they were fed *Bifidobacterium breve*. Sterile water was used as the control group. Each group consisted of three sections, with 120 silkworm larvae per section. They were fed normal mulberry leaves until day 4 of the 5th instar. From day 4 of the 5th instar, they were fed mulberry leaves containing trace amounts of pesticide residues (premature feeding led to pesticide poisoning symptoms in the larvae). The number of poisoned silkworm larvae in each experimental group was counted, and their survival rate was statistically analyzed. The mulberry leaves containing trace amounts of pesticide residues were those affected by aerial pesticide spraying, primarily containing organophosphate insecticides. Symptoms of pesticide poisoning in silkworm larvae included curling, spitting out water, and failing to spin cocoons 1-3 days after being fed mulberry leaves containing trace amounts of pesticide residues.

[0089] Statistical results are as follows Figure 9 As shown, compared with the control group (CK) fed with blank culture medium, the larval viability of the two probiotic-fed groups was significantly improved. The cocooning rate of the Lactobacillus rhamnosus group (LR) reached 78.59%, significantly higher than that of the Bifidobacterium breve group (Bb) and the control group (CK) (P < 0.05). This indicates that supplementing with Lactobacillus rhamnosus can improve the viability of silkworm larvae and reduce pesticide poisoning.

[0090] Example 11

[0091] The silkworms were fed *Lactobacillus rhamnosus* according to the method in Example 2 and *Bifidobacterium breve* according to the method in Comparative Example 4. Sterile water was sprayed as a control group. Each group consisted of three sections, with 120 silkworm larvae in each section. They were fed normal mulberry leaf rearing until they reached maturity and spun cocoons. The pupae, after four days of pupation, were freeze-dried, and their basic nutrient composition was determined. The determination methods were as follows: crude protein content was determined using the Kjeldahl method according to GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food"; fat content was determined using the Soxhlet extraction method according to GB5009.6-2016 "National Food Safety Standard - Determination of Fat in Food"; and total sugar content was determined according to GB / T 9695.31-2008 "Determination of Total Sugar Content in Meat Products".

[0092] The results are shown in Table 1: The crude protein content of the Lactobacillus rhamnosus group was significantly higher than that of the control group and the Bifidobacterium breve group (P<0.05), and the crude fat content was significantly lower than that of the Bifidobacterium breve group (P<0.05), showing a decreasing trend compared with the control group. The two probiotic-added groups significantly reduced the total sugar content (P<0.05).

[0093] Table 1. Basic nutrient analysis of silkworm pupae after feeding with different probiotics.

[0094] detection indicators CK Lr Bb Crude protein / % 64.536±0.465b 67.638±0.281a 61.084±0.356c Crude fat / % 24.002±0.058ab 23.145±0.969b 25.583±0.325a Total sugar / % 5.076±0.02a 4.411±0.062b 4.166±0.088b

[0095] Note: In the table, CK, LR and Bb represent the control group, Lactobacillus rhamnosus group and Bifidobacterium breve group, respectively. Lowercase letters in the same row indicate significant differences among different feeding groups (p < 0.05).

[0096] Example 12

[0097] The silkworms were fed with *Lactobacillus rhamnosus* as in Example 2 and *Bifidobacterium breve* as in Comparative Example 4, with sterile water sprayed as a control group. Each group consisted of three sections, with 120 silkworm larvae in each section. They were fed with normal mulberry leaves until they became mature silkworms and spun cocoons. After the silkworm pupae had been pupated for 4 days, they were freeze-dried, and crude fat was extracted from the silkworm pupae according to the crude fat determination method in Example 7. Then, oil samples were taken for methyl esterification and GC-MS analysis.

[0098] The results are as follows: Figure 10 As shown, the content of saturated fatty acids in silkworm pupae fed with Lactobacillus rhamnosus was significantly lower, while the content of unsaturated fatty acids, ω-3 fatty acids and essential fatty acids was significantly higher than that in the control group and the Bifidobacterium breve group (P < 0.05).

[0099] 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. A method for improving the growth and nutritional traits of silkworms, characterized in that, Includes the following steps: When silkworm larvae are raised to the fourth instar, they are fed with Lactobacillus rhamnosus. The dosage of Lactobacillus rhamnosus is 0.05-0.1 mL per fourth instar silkworm. And / or, when silkworm larvae are raised to the fifth instar, they are fed with Lactobacillus rhamnosus, and the dosage of Lactobacillus rhamnosus is 0.1-0.2 mL / silkworm for the fifth instar. The viable count of Lactobacillus rhamnosus per milliliter is 10. 6 -10 8 CFU / mL; The Latin name of the Lactobacillus rhamnosus is Lactobacillus rhamnosus, and its accession number is ATCC53103.

2. The method according to claim 1, characterized in that, The method for adding Lactobacillus rhamnosus is as follows: spray Lactobacillus rhamnosus bacterial solution onto the surface of mulberry leaves.

3. The method according to claim 1, characterized in that, The method for preparing the Lactobacillus rhamnosus includes the following steps: Lactobacillus rhamnosus was activated and cultured to obtain Lactobacillus rhamnosus bacterial solution; The Lactobacillus rhamnosus bacterial solution was diluted to obtain a diluted Lactobacillus rhamnosus bacterial solution; Mulberry oligosaccharides and Tween-80 were added to the diluted Lactobacillus rhamnosus bacterial solution for later use.

4. The method according to claim 3, characterized in that, The culture medium for the Lactobacillus rhamnosus is MRS medium.

5. The method according to claim 3, characterized in that, The culture conditions for the *Lactobacillus rhamnosus* were 32-37°C, with shaking at 180 rpm overnight.

6. The method according to claim 3, characterized in that, Dilute the Lactobacillus rhamnosus bacterial suspension to 10. 6 -10 8 CFU / mL.

7. The method according to claim 3, characterized in that, The amount of mulberry oligosaccharide added to the diluted Lactobacillus rhamnosus bacterial solution is 0.4-0.6 g / 100 mL; the amount of Tween-80 added is 0.1-0.5 ml / 100 mL.

8. The application of the method according to any one of claims 1 to 7 in the cultivation of high-quality edible silkworm pupae.

9. The application of the method according to any one of claims 1 to 7 in the prevention and control of silkworm diseases.

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