Method for rapidly screening blood sugar reducing probiotics based on silkworm model

Abstract

A method for rapidly screening blood sugar reducing probiotics based on a bombyx mori model comprises the following steps: fasting bombyx mori bred to 5 years old overnight, then feeding the bombyx mori with high-sugar-content mulberry leaves, continuously feeding the bombyx mori with probiotics after 1.5-2.5 hours, detecting the blood sugar level of the bombyx mori after 2-3 hours, and screening out a probiotic strain with a remarkable blood sugar reducing effect through contrastive analysis. According to the method, the silkworms are fed with the specific sugar-containing mulberry leaves, a specific instantaneous high-glucose model is established, the probiotics capable of achieving the blood glucose reducing effect in the organism can be effectively screened out, the method is short in time consumption period, low in cost and high in precision, a large number of experiments can be conducted in a short time, and the method is suitable for large-scale popularization and application. The blood sugar reducing probiotics which still have the blood sugar reducing effect after entering the body are quickly screened out. The effectiveness is further verified in combination with a diabetic mouse model, the hypoglycemic mechanism of the probiotics is researched, and a theoretical basis is provided for subsequent application of the probiotics.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic screening and application, and in particular to a method for quickly screening blood sugar-lowering probiotics based on a silkworm model. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by insulin resistance and impaired insulin secretion. Insulin is an effective treatment drug, but it is expensive. Although there are many types of other glucose-lowering drugs, each has side effects, such as hypoglycemia and obesity. Therefore, a safe method or drug with no toxic side effects is urgently needed to treat T2DM.

[0003] It has been found in the prior art that some probiotics may be a promising auxiliary treatment for patients with T2DM. In recent years, both experimental and clinical progress has been made. However, due to the complexity and differences in the hypoglycemic mechanisms of different probiotics, some probiotics have good results in in vitro tests, but the effects after entering the body are not ideal. At present, the probiotics that have been widely studied for hypoglycemic effects are mostly lactobacilli and bifidobacteria, and many other genera have not been discovered. Therefore, it is still necessary to continue to screen probiotics with hypoglycemic potential, good stability in the intestine and strong safety.

[0004] Currently, mammals such as mice and rats have been widely used to screen anti-diabetic drugs, but the high cost of maintaining a large number of animals and ethical issues surrounding animal welfare are serious disadvantages of these models. In order to overcome the high cost and ethical issues of using mammalian models for drug development, the existing technology uses silkworm models to screen acute anti-diabetic drugs. However, this process requires long-term high-sugar feeding to build silkworms into a stable T2DM model in advance, otherwise it cannot play a screening role for all hypoglycemic drugs, as recorded in the literature "Evaluation of anti-diabetic drugs by usingsilkworm, Bombyx mori Yasuhiko Matsumoto, Kazuhisa Sekimizu *".

[0005] Due to the short cycle time of the silkworm diabetes model and its own metabolic properties, the T2DM model has a short stability time and unstable fluctuations in blood sugar levels, which cannot be used as a good model for screening blood sugar-lowering probiotics. Summary of the invention

[0006] Based on the above technical problems, the present invention aims to provide a method for rapid screening of hypoglycemic probiotics based on a silkworm model. Through a specific feeding method, the hypoglycemic probiotics are screened by using the transient high sugar produced in the silkworm body. This method has a short cycle, low cost, high accuracy, and can perform a large number of experiments in a short period of time, so as to quickly screen out hypoglycemic probiotics that still have the effect of lowering blood sugar after entering the body.

[0007] The purpose of the present invention is achieved through the following technical solutions: A rapid screening method for hypoglycemic probiotics based on a silkworm model, characterized in that: silkworms cultured to 5 years old are fasted overnight, and then mulberry leaves containing high sugar are fed to the silkworms, and mulberry leaves containing probiotics are continued to be fed after an interval of 1.5 to 2.5 hours, and the blood sugar level of the silkworms is detected 2 hours after feeding the mulberry leaves, and probiotic strains with significant hypoglycemic effects are screened out through comparative analysis.

[0008] Furthermore, the comparative analysis is based on the silkworm group fed with mulberry leaves containing probiotics as the bacteria-feeding group, and a blank group, a high-sugar group, a control group and the bacteria-feeding group are set up for blood sugar content detection and comparative analysis, wherein each group is as follows: Blank group: silkworms were fasted overnight, then fed with ordinary mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 h later; High sugar group: silkworms were fasted overnight, then fed with sugary mulberry leaves, and then continued to be fed with sugary mulberry leaves 1.5-2.5 hours later; Control group: silkworms were fasted overnight, then fed with sugar-containing mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 hours later; Bacteria-fed group: The silkworms were fasted overnight, then fed with sugar-containing mulberry leaves, and then fed with bacteria-containing mulberry leaves 1.5-2.5 hours later; If the blood sugar level of the fed bacteria group is lower than that of the control group, it means that the probiotics are the selected blood sugar-lowering probiotics.

[0009] Furthermore, the high-sugar mulberry leaves are prepared by spraying a sucrose solution with a mass concentration of 10-18% evenly on the surface of the mulberry leaves, and collecting the leaves after they are naturally dried, with a spraying amount of 10 mL / 15 g of mulberry leaves.

[0010] Furthermore, the mulberry leaves containing probiotics are prepared by spraying skim milk with a mass concentration of 12-16% on the surface of the mulberry leaves, wherein the skim milk contains probiotics, and the probiotic content is 10 9 CFU / mL, the appropriate amount of spray is 2mL / 10g mulberry leaves.

[0011] The blank mulberry leaves are prepared by directly spraying skim milk with a mass concentration of 12-16% on the surface of the mulberry leaves, and then naturally drying and collecting.

[0012] When using silkworms to prepare a high-sugar model, due to the metabolic characteristics of the silkworms themselves, we found that in the process of long-term feeding of silkworms to construct a diabetes model, the blood sugar changes in the silkworms became unstable as the culture time prolonged. When the mature diabetic silkworm model was used to screen blood sugar-lowering probiotics, it was difficult for the probiotics to take effect quickly, and the purpose of rapid screening could not be achieved. Therefore, we tried to establish an instantaneous high-sugar silkworm model, but in the process of establishing the instantaneous high-sugar model, we found that specific feeding conditions were required for the instantaneous high-sugar physiological state of silkworms to be close to the pre-diabetes or mild diabetes state of humans, in order to simulate the human body's response mechanism to blood sugar regulation and screen out probiotics that have a blood sugar-lowering effect on the human body. If the feeding conditions are not suitable, even if the silkworms develop a high-sugar state, the blood sugar concentration value and the changes in the blood sugar concentration value at different subsequent test intervals cannot be controlled, and it is impossible to accurately screen out blood sugar-lowering probiotics.

[0013] In the present invention, the sugar-containing mulberry leaves are prepared by using a sucrose solution with a concentration of 10-18% to raise silkworms, and the bacteria-containing mulberry leaves are fed again after a specific interval of 1.5-2.5 h. If the concentration / spraying of the sucrose solution is relatively low, after the silkworms produce instantaneous high sugar, they will be rapidly metabolized by the silkworms themselves, and it is impossible to maintain a high sugar state for a period of time for subsequent feeding of bacteria-containing mulberry leaves. The thalline takes effect in the silkworm body, that is, the probiotics have not taken effect, and the blood sugar concentration can return to normal under the metabolism of the silkworm itself. However, the sucrose solution concentration / spraying amount is too high, and the interval time of feeding bacteria-containing mulberry leaves is too short or too long, and the blood sugar value in the silkworm body is not compatible with the probiotic effect, which will cause the accuracy of the screened probiotics to be unbalanced, and some probiotics that may not be significantly effective will also be screened.

[0014] Further, the 5-year-old silkworms are cultured according to the following steps: (1) Accelerating the growth of silkworms and collecting larvae: Place silkworm eggs on a ventilated bamboo cage and maintain the culture temperature at 22°C. Place them in an incubator and maintain the incubator temperature at 25°C. Accelerate the growth of silkworm eggs until 20% of the silkworm eggs on a sheet are green. Wrap the silkworm eggs with the egg surface facing down and shade them from light to make them black so that the eggs develop uniformly. Wait until the larvae hatch and collect the larvae. (2) Breeding of young silkworms: The temperature for the first-stage silkworms is 27-28°C, the relative humidity is 95%, and they are fed with light green mulberry leaves; the temperature for the second-stage silkworms is 26-27°C, the relative humidity is 85-90%, and they are fed with dark green leaves; the temperature for the third-stage silkworms is 25-26°C, the relative humidity is 80-85%, and they are fed with three green leaves; (3) Silkworm breeding: During the 4th instar period, the breeding temperature is maintained at 25°C and the relative humidity is maintained at 60-70%. Ventilation is maintained until the silkworms wake up from their 5th instar hibernation. They can then be used for subsequent high-sugar model establishment and probiotic screening experiments.

[0015] The present invention has the following technical effects: In the present invention, by feeding silkworms with specific sugar-containing mulberry leaves, a specific instantaneous high-sugar model is established, which can effectively screen out probiotics that can play a hypoglycemic effect in the body. This method has a short cycle time, low cost, high accuracy, and can carry out a large number of experiments in a short period of time, so as to quickly screen out hypoglycemic probiotics that still have a hypoglycemic effect after entering the body. Its effectiveness is further verified in combination with a diabetic mouse model, and the hypoglycemic mechanism of probiotics is studied to provide a theoretical basis for its subsequent application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Bar graph showing the inhibitory effect of α-glucosidase in vitro by 10 probiotic strains.

[0017] Figure 2 :Results of hemolytic activity test of 9 probiotic strains.

[0018] Figure 3 : Bar graph of blood sugar changes in different strains of silkworm models for screening hypoglycemic probiotics.

[0019] Figure 4 : Bar graph of random blood glucose and fasting blood glucose changes in mouse experiments.

[0020] Figure 5 : Graph of the results of the glucose tolerance test in the small number experiment. DETAILED DESCRIPTION

[0021] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0022] The present invention involves screening of 10 probiotic strains, which are numbered XJ810, XZ306, XJ335, XZ4410, XZ106, XJ243, XJ244, XJ402, XZ2010 and XZ502, and are identified as Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus acidilactici, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus mucosa reuteri, Weissella fusion and Leuconostoc pseudomesenteroides through sequence comparison.

[0023] Example 1 In vitro screening of probiotics for lowering blood sugar: 100 μL of the preserved bacterial solution was activated in MRS liquid medium for two generations and then used for extraction of cell-free supernatant: the test bacterial solution was inoculated into MRS medium at a ratio of 2% and cultured at 37 °C for 24 h, followed by centrifugation at 6000 r / min for 10 min, the supernatant was collected and filtered through a 0.22 μm microporous filter membrane to obtain the cell-free supernatant (CFS).

[0024] α-Glucosidase inhibition rate determination: For each of the above strains, 25 μL of 0.1 mol / L PBS buffer (pH 6.8) was first added to the wells of a 96-well plate, followed by 25 μL of 20 mmol / L PNPG and 25 μL of sample; incubated at 37°C for 10 min, and then 50 μL of 0.2U / mL α-glucosidase was added; incubated at 37°C for 15 min, and finally 100 μL of 0.1 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance (A) of the reaction solution at a wavelength of 405 nm was detected using an ELISA reader. Using 1 mg / mL acarbose as a positive control, the inhibition rate is shown as follows: ×100 In the formula: blank is PBS+PNPG+PBS+enzyme+NA2CO3; blank control is PBS+PNPG+PBS+ PBS +NA2CO3; sample is PBS+PNPG+sample+enzyme+NA2CO3; sample control is PBS+PNPG+sample+PBS +NA2CO3.

[0025] The α-glucosidase inhibition rates of strains XZ106, XZ502, XJ810, XZ2010, XJ243, XJ244, XZ306, XJ335, XJ402, and XZ4410 were 14.59±1.31, 9.83±2.31, 31.69±4.16, 13.56±4.45, 10.87±1.52, 10.5±1.74, 10.47±1.76, 6.97±1.72, 3.82±1.08, and 7.12±2.19, respectively. The α-glucosidase inhibition rates measured were as follows: Figure 1 XJ402 was excluded based on the in vitro glucose-lowering index, and the safety of the remaining 9 lactic acid bacteria was evaluated.

[0026] Safety Testing: (1) Hemolytic test The activated probiotics were streaked on Columbia blood agar plates, and the pathogenic strain Staphylococcus aureus SD was inoculated as a control at the same time. The plates were placed at 37°C for 48 hours. Finally, hemolysis was observed around the colonies and photographed. The results were as follows: Figure 2 shown.

[0027] As can be seen from the figure, Staphylococcus aureus showed obvious β-hemolysis, while strains XJ810, XJ243, XJ244 and XZ4410 had certain hemolysis, and strains XZ106, XZ502, XZ2010, XZ306, and XJ335 had no obvious hemolysis.

[0028] Therefore, when we subsequently used the silkworm model for preliminary screening, we only used 5 probiotic strains that did not have hemolysis for preliminary screening tests. In the in vitro blood sugar lowering test results of these 5 probiotic strains, the blood sugar lowering effects were XZ106>XZ2010>XZ306>XZ502>XJ335.

[0029] Example 2 Rapid screening of hypoglycemic probiotics using the silkworm model: 1. Raise silkworms from fertilization to the fifth instar. The specific steps are as follows: (1) Accelerating the growth of silkworms and collecting silkworm eggs: Place silkworm eggs (of the Sichuan, Shan, and Shushui varieties) on ventilated bamboo cages and maintain a culture temperature of 22°C. Place them in an incubator and maintain an incubator temperature of 25°C. Accelerate the growth of silkworm eggs until 20% of the silkworm eggs on a sheet of silkworm eggs are green. Wrap the silkworm eggs with the egg surface facing downward and shade them to make them black so that the eggs develop uniformly. From the day of greening, the egg surface will turn gray on the second day. On the third day, the silkworms will hatch. Finally, collect the silkworm eggs. Sprinkle finely chopped fresh mulberry leaves on the silkworm seed paper. After the silkworms crawl onto the mulberry leaves, pour the silkworms and the mulberry leaves on the silkworm bed paper and spread the silkworms evenly. (2) Breeding of young silkworms: The temperature for the first-stage silkworms is 27-28°C, the relative humidity is 95%, and they are fed with light green mulberry leaves; the temperature for the second-stage silkworms is 26-27°C, the relative humidity is 85-90%, and they are fed with dark green leaves; the temperature for the third-stage silkworms is 25-26°C, the relative humidity is 80-85%, and they are fed with three green leaves; (3) Silkworm breeding: During the 4th instar period, the breeding temperature is maintained at 25°C and the relative humidity is maintained at 60-70%. Ventilation is maintained until the silkworms wake up from their 5th instar hibernation. They can then be used for subsequent high-sugar model establishment and probiotic screening experiments.

[0030] 2. A high-sugar model was established for the 5-year-old silkworms to conduct a rapid initial screening of blood sugar-lowering probiotics. The specific screening steps are as follows: The silkworms that woke up from the fifth instar were divided equally into four groups; Blank group: silkworms were fasted overnight, then fed with ordinary mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 h later; High sugar group: silkworms were fasted overnight, then fed with sugary mulberry leaves, and then continued to be fed with sugary mulberry leaves 1.5-2.5 hours later; Control group: silkworms were fasted overnight, then fed with sugar-containing mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 hours later; Bacteria-fed group: The silkworms were fasted overnight and then fed with sugar-containing mulberry leaves. 1.5 to 2.5 hours later, they were fed with bacteria-containing mulberry leaves. Five different bacteria-fed groups were prepared according to the five strains.

[0031] The high-sugar mulberry leaves are prepared by spraying a sucrose solution with a mass concentration of 10-18% evenly on the surface of the mulberry leaves, and then collecting the leaves after they are naturally dried. The spraying amount is 10 mL / 15 g of mulberry leaves.

[0032] The mulberry leaves containing probiotics are prepared by spraying skim milk with a mass concentration of 12-16% on the surface of the mulberry leaves, wherein the skim milk contains probiotics with a probiotic content of 10 9 CFU / mL, the appropriate amount of spray is 2mL / 10g mulberry leaves.

[0033] The blank mulberry leaves are obtained by directly spraying skim milk with a mass concentration of 12-16% on the surface of the mulberry leaves, and then naturally drying and collecting them. The ordinary mulberry leaves are fresh mulberry leaves that are sprayed with any liquid.

[0034] After feeding mulberry leaves for 2 hours, the blood sugar level in the blood sugar level was measured by blood glucose meter every 1 hour. Figure 3 As shown in the figure. As can be seen from the bar graph, for the Sichuan-Shan-Shu-Shui variety silkworm, only three strains with hypoglycemic effect screened in vitro have hypoglycemic effect on the silkworm model, namely XZ106, XZ306 and XJ335. The 871 and Liangguang No. 2 varieties of silkworms were used again to further verify the effectiveness of the model established with Shanchuan-Shu-Shui, and the results were consistent with the screening results of the above-mentioned mountain-water variety silkworm model.

[0035] From the comparison between Example 1 and Example 2, it can be seen that among the probiotics screened by in vitro primary screening, XJ335 is the strain with the worst effect in the in vitro hypoglycemic test results, among which XZ2010 has a significantly better hypoglycemic effect in vitro than XZ306 and XJ335, but the results of the silkworm model test show that XZ2010 has the worst hypoglycemic effect. This further illustrates that the hypoglycemic mechanism of probiotics in vivo is more complicated than that of chemical drugs. In addition to inhibiting glycosidase activity, there are many different pathways of action, which also affect each other, resulting in strains with excellent glycosidase inhibition in vitro. Activity may not play a hypoglycemic effect in vivo, such as XZ2010, and probiotics like XJ335 do not achieve hypoglycemic effects by inhibiting glycosidase activity in vivo, but promote hypoglycemia through other complex unknown mechanisms.

[0036] Example 3 In order to further verify the accuracy of the rapid initial screening of hypoglycemic probiotics based on the silkworm model, the mouse diabetes model was further used to verify the effect of the screened hypoglycemic strains: The strains were cultured in MRS broth at 37 °C for 18 h. The cells were harvested by centrifugation at 6500 g for 5 min at 4 °C. The collected cells were washed twice with sterile PBS and adjusted to 5 × 10 9 CFU mL−1. Freshly prepared bacterial suspension was administered by gavage daily, 200 μL per mouse. Mice were randomly divided into 2 groups; the group of 6 mice on a normal diet served as the control group (control). The other mice were fed HFD to establish the T2DM model. After six weeks of control feeding, HFD-fed mice were fasted for 12 h and injected intraperitoneally with STZ (streptozotocin) dissolved in citrate buffer (pH 4.5) at a dose corresponding to 50 mg / kg body weight (BW) for three consecutive days. The control group was injected with an equal amount of citrate buffer. Blood from the tail vein was taken to measure the fasting blood glucose (FBG) level, and FBG levels ≥ 11.1 mmol / L indicated that the T2 DM mouse model was successfully established. Diabetic mice were randomly divided into 4 groups (n = 6): diabetic model group (Model), positive control group (Metformin, 150 mg / kgBW), XZ106 group (XZ106), XZ306 group (XZ306), XJ335 (XJ306), XZ2010 group (XZ2010) and XZ502 group (XZ502), and the corresponding preparations were gavaged once a day. Sterile water was given to the normal group and the diabetic group. The mice in the normal group were fed with ordinary food, and the mice in the other groups continued to be fed with high-fat diet. Random blood glucose (RBG) and fasting blood glucose (FBG) were recorded weekly, and the glucose tolerance of the mice was measured in the last week.

[0037] The results of random blood glucose and fasting blood glucose of mice every week and fasting glucose tolerance before sacrifice were as follows Figure 4 and Figure 5 As shown in the figure. As can be seen from the figure, after the bacteria are given, the blood sugar of mice in the XZ106, XZ306 and XJ335 groups has a tendency to be suppressed or reduced, and according to the results of glucose tolerance, the hyperglycemia symptoms of the three groups of mice given bacteria corresponding to XZ106, XZ306 and XJ335 are alleviated, while the hyperglycemia symptom relief effect of ZX2010 and ZX502 for small numbers is not obvious. The reliability of the hypoglycemic probiotic effect obtained by the initial screening of the silkworm model is further verified. That is, the present invention realizes the rapid screening of hypoglycemic probiotics by setting up the silkworm instantaneous high sugar model, effectively saves the screening time, and the screening result has a high degree of accuracy.

Claims

1. A rapid screening method for hypoglycemic probiotics based on a silkworm model, characterized in that: Silkworms raised to the fifth age were fasted overnight and then fed with mulberry leaves containing high sugar. After an interval of 1.5 to 2.5 hours, they were continued to be fed with mulberry leaves containing probiotics. The blood sugar levels of the silkworms were tested 2 hours after feeding the mulberry leaves. Through comparative analysis, the probiotic strains with significant blood sugar-lowering effects were screened out.

2. The method for rapid screening of blood sugar-lowering probiotics based on a silkworm model according to claim 1, characterized in that: The comparative analysis is based on the silkworm group fed with mulberry leaves containing probiotics as the bacteria-feeding group, and a blank group, a high-sugar group, a control group and the bacteria-feeding group are set up for blood sugar content detection and comparative analysis, wherein each group is as follows: Blank group: silkworms were fasted overnight, then fed with ordinary mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 h later; High sugar group: silkworms were fasted overnight, then fed with sugary mulberry leaves, and then continued to be fed with sugary mulberry leaves 1.5-2.5 hours later; Control group: silkworms were fasted overnight, then fed with sugar-containing mulberry leaves, and then fed with blank mulberry leaves 1.5-2.5 hours later; Bacteria-fed group: The silkworms were fasted overnight, then fed with sugar-containing mulberry leaves, and then fed with bacteria-containing mulberry leaves 1.5-2.5 hours later; If the blood sugar level of the fed bacteria group is lower than that of the control group, it means that the probiotics are the selected blood sugar-lowering probiotics.

3. A rapid screening method for hypoglycemic probiotics based on a silkworm model as claimed in claim 1 or 2, characterized in that: The high-sugar mulberry leaves are prepared by spraying a sucrose solution with a mass concentration of 10-18% evenly on the surface of the mulberry leaves, and then collecting the leaves after they are naturally dried. The spraying amount is 10 mL / 15 g of mulberry leaves.

4. A method for rapid screening of blood sugar-lowering probiotics based on a silkworm model according to any one of claims 1 to 3, characterized in that: The mulberry leaves containing probiotics are prepared by spraying skim milk with a mass concentration of 12-16% on the surface of the mulberry leaves, wherein the skim milk contains probiotics with a probiotic content of 10 9 CFU / mL, the appropriate amount of spray is 2mL / 10g mulberry leaves.

5. A method for rapid screening of blood sugar-lowering probiotics based on a silkworm model according to any one of claims 1 to 4, characterized in that: The 5-year-old silkworm is cultured according to the following steps: (1) Accelerating the growth of silkworms and collecting larvae: Place silkworm eggs on a ventilated bamboo cage and maintain the culture temperature at 22°C. Place them in an incubator and maintain the incubator temperature at 25°C. Accelerate the growth of silkworm eggs until 20% of the silkworm eggs on a sheet are green. Wrap the silkworm eggs with the egg surface facing down and shade them from light to make them black so that the eggs develop uniformly. Wait until the larvae hatch and collect the larvae. (2) Breeding of young silkworms: The temperature for the first-stage silkworms is 27-28°C, the relative humidity is 95%, and they are fed with light green mulberry leaves; the temperature for the second-stage silkworms is 26-27°C, the relative humidity is 85-90%, and they are fed with dark green leaves; the temperature for the third-stage silkworms is 25-26°C, the relative humidity is 80-85%, and they are fed with three green leaves; (3) Silkworm breeding: During the 4th instar period, the breeding temperature is maintained at 25°C and the relative humidity is maintained at 60-70%. Ventilation is maintained until the silkworms wake up from their 5th instar hibernation. They can then be used for subsequent high-sugar model establishment and probiotic screening experiments.

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