Breeding method of medicinal stiff pupae

Abstract

The invention provides a method for breeding medicinal stiff pupae, which is a dry pupa body obtained after the pupae of Bombyx mori Linnaeus is inoculated with Beauveria bassiana (Balls.) Vuilant to culture and breed, and comprises the following steps of: a, breaking cocoons and taking the pupae, and sterilizing by using 75% ethanol; b, performing spray inoculation for 2-3 times at the concentration of 1 * 107-5 * 107 pieces / ml, wherein the inoculation density is preferably 3000 pieces / m < 2 >-4000 pieces / m < 2 >; and c, culturing in an environment with the temperature of 23-28 DEG C and the relative humidity of 75-85% for 7-10 days to obtain the strain. According to the method, live pupae are used as inoculation objects, the integrity of pupa bodies is guaranteed, and the loss of medicinal components is avoided. According to the preparation process, the concentration of the beauveria bassiana liquid, the inoculation frequency and the culture conditions are accurately controlled, the growth of the beauveria bassiana in the pupa body is ensured, and the content of the beauveria bassiana is ensured. Compared with a literature method, the pretreatment method is simple in process, labor cost is saved, silkworm chrysalis death is avoided while sterility is guaranteed, silkworm chrysalis vitality can be weakened, and the dip dyeing capacity of beauveria bassiana can be improved. The preparation conditions are accurately controlled, the quality of the stiff pupae is improved, the production process is simplified, and large-scale popularization and application are easy.

Description

Technical Field

[0001] The invention relates to a method for cultivating medicinal pupae. Background Art

[0002] Dead pupae are derived from the dried pupae of the silkworm Bombyx mori Linnaeus, an insect of the Bombyceae family, inoculated with the fungus Beauveria bassiana (Bals.) Vuillant. They possess sedative, antispasmodic, and antitussive properties and are used to treat epilepsy, febrile convulsions, enuresis, diabetes, and chronic tracheitis. The Chinese patent medicines marketed using dead pupae as raw materials include Tiancan Tablets and Tiancan Capsules, but both face raw material shortages and insufficient supply. The underlying reason for this is that existing literature on dead pupae preparation is simplistic and lacks key cultivation techniques.

[0003] Liu Xuexiang et al., Research on the Improvement of the Fermentation Method of Silkworm Pupae, Chinese Journal of Medicinal Biotechnology, 10(04): 356-360. The fermentation method of silkworm pupae was improved. 10.0 g of silkworm pupa powder was weighed and moistened with the powder at a material-water ratio of 1:1. The mixture was filled into a test tube and mixed evenly. The tube mouth was covered with plastic paper and tied with string. The tube was sterilized at 121°C for 30 minutes. After cooling, the mixture was used as the fermentation medium. Liquid strains of Beauveria bassiana were inoculated under sterile conditions and cultured in an incubator at (27±1)°C. The growth was observed and recorded regularly. After 15 days of fermentation, the medium was removed until mycelium grew inside and outside the medium. The mixture was dried at 60°C for 24 hours and passed through an 80-mesh sieve.

[0004] Wang Chenhao et al., "Effects of Three Surfactants on the Preparation of Bombyx Batryticatus," Journal of Traditional Chinese Medicine, Vol. 40, No. 5, May 2018. The effects of three surfactants on the preparation of bombyx batryticatus were investigated. Live and dead silkworm pupae were used as fermentation substrates and inoculated with Beauveria bassiana spores isolated from local Bombyx batryticatus in Panzhihua, Sichuan. The effects of different surfactants on the preparation process were investigated. The bombyx batryticatus pupae were tested according to the test items for Bombyx batryticatus in Part I of the Chinese Pharmacopoeia (2015), and their quality was compared with that of commercially available Bombyx batryticatus.

[0005] Liu Xuexiang et al., "Study on Improved Fermentation Methods for Silkworm Pupae," Chinese Journal of Medicinal Biotechnology, Vol. 10, No. 4, August 2015. Silkworm pupae were used as the fermentation substrate, inoculated with a liquid culture of Beauveria bassiana to produce silkworm pupae. The effects of different inoculum sizes on the fermentation process were studied, with ammonium oxalate content as the primary indicator, combined with substrate conversion rate. The results were compared with the ammonium oxalate content in the herbal medicinal material Bombyx batryticatus.

[0006] Song Jianmin, et al., Determination of the anti-convulsion active ingredients of white muscardine pupae instead of white silkworm pupae, Journal of Hefei University (Natural Science Edition), Vol. 14, No. 2, June 2004. In the experiment of measuring the anti-convulsion active ingredients of silkworm pupae by oscillographic titration potentiometer method with live pupae as the control, the white muscardine pupae generated by inoculation with dead pupae had the lowest ammonium oxalate content, and the difference reached a significant level; while the white muscardine pupae generated by inoculation with live pupae had similar ammonium oxalate contents to those generated by inoculation with larvae, and the difference was not significant; the white muscardine pupae generated by puncturing live pupae had the highest ammonium oxalate content, and the difference was significant.

[0007] Existing methods for fermenting pupae using silkworm pupa powder or dead silkworm pupae often involve inoculating Beauveria bassiana culture fluid for cultivation. However, existing methods cannot guarantee the integrity of the pupae. "Distinguishing appearance from quality" is the essence of traditional Chinese medicine identification. Pupae prepared using silkworm pupa powder lack complete appearance characteristics. First, their quality cannot be judged by their appearance. Second, because Chinese medicinal materials generally have similar appearances after pulverization, the prepared pupae powder increases the difficulty of identification and is prone to counterfeiting. Using dead pupae as inoculation targets to prepare pupae cannot guarantee the beauvericin content, and the puncture inoculation process is complex, making it difficult to apply industrially. Furthermore, existing fermentation processes lack precise control over the concentration of Beauveria bassiana culture fluid, which can easily affect the quality of the medicinal material. Using ammonium oxalate as a quality control indicator for pupae in finished products lacks specificity, and ammonium oxalate is a significant irritant, which can easily cause multiple negative effects on human health. Summary of the Invention

[0008] The invention provides a method for cultivating medicinal pupae.

[0009] The present invention provides a method for cultivating medicinal pupae, which is prepared by inoculating pupae of silkworms, Bombyx mori Linnaeus, an insect of the Bombyceae family, with Beauveria bassiana (Bals.) Vuillant for cultivation and reproduction, and comprises the following steps:

[0010] a. Breaking the cocoon and taking out the pupa, and disinfecting it with 75% ethanol; the pupa is a living pupa;

[0011] b. With a concentration of 1*10 7 -5*10 7 Spray inoculation 2-3 times, with an inoculation density of 3000 cells / m 2 ~4000 pieces / m 2 appropriate;

[0012] c. Place it in an environment with a temperature of 23-28℃ and a relative humidity of 75-85% and cultivate it for 7-10 days.

[0013] Wherein, the inoculation concentration described in step b is 5*10 7 / ml; the spray inoculation frequency was 3 times.

[0014] The inoculation density is 3000 cells / m 2 .

[0015] c The culture time is 10 days.

[0016] The present invention uses live pupae as inoculation targets, ensuring the integrity of the pupae and avoiding loss of active ingredients. Simultaneously, its preparation process precisely controls the concentration of the Beauveria bassiana liquid, the number of inoculations, and the culture conditions, ensuring the growth of Beauveria bassiana within the pupae and the content of beauvericin. Compared to methods described in the literature, its pretreatment method is simpler, saves labor costs, ensures sterility without killing the silkworm pupae, and can also weaken the pupae's vitality, thereby increasing the infectivity of Beauveria bassiana. The present invention's precise control of its preparation conditions improves the quality of the pupae, simplifies the production process, and facilitates large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 5*10 6 / mL inoculation;

[0018] Figure 2 10 7 / mL inoculation;

[0019] Figure 3 5*10 7 / mL inoculation;

[0020] Figure 4 10 8 / mL inoculation;

[0021] Figure 5 Live pupal inoculation;

[0022] Figure 6 dead pupae inoculation;

[0023] Figure 7 Infiltration 5*10 7 / mL;

[0024] Figure 8 Spray 5*10 7 / mL;

[0025] Figure 9 The silkworm pupae turn black and suppurate;

[0026] Figure 10 Spray 5*10 6 / mL;

[0027] Figure 11 Spray 5*10 7 / mL;

[0028] Figure 12Spray 5*10 8 Pieces / ml;

[0029] Figure 13 Culture for 7 days;

[0030] Figure 14 Culture for 8 days;

[0031] Figure 15 Culture for 9 days;

[0032] Figure 16 Culture for 10 days;

[0033] Figure 17 Vaccination 2 times;

[0034] Figure 18 Vaccination 3 times;

[0035] Figure 19 Vaccination 4 times;

[0036] Figure 20 Method 1: preparing stiff pupae;

[0037] Figure 21 Method 2: preparing stiff pupae;

[0038] Figure 22 Method 3: preparing stiff pupae;

[0039] Figure 23 Method 4: preparing stiff pupae;

[0040] Figure 24 Determination of beauvericin content (from bottom to top: reference substance, method 2, method 3, method 4, present invention, present invention). DETAILED DESCRIPTION

[0041] Example 1: Method for cultivating medicinal pupae of the present invention

[0042] After the cocoons were broken and the pupae were taken out, they were disinfected with 75% ethanol and the concentration of 5*10 7 Spray inoculation was performed 3 times with a density of 3000 cells / m 2 ~4000 pieces / m 2 It is best to place it in an environment with a temperature of 23-28℃ and a relative humidity of 75-85% and cultivate it for 10 days.

[0043] Example 2: Steps and parameter screening test of the cultivation method of medicinal pupae of the present invention

[0044] 1Inspection of vaccination subjects

[0045] 1.1 Inoculation of adult silkworms

[0046] Spray inoculation was performed on the 6th and 7th days of the 5th instar silkworm, and the inoculation concentration of Beauveria bassiana was set at 5*106 , 10 7 , 5*10 7 , 10 8 pieces / mL.

[0047] After being inoculated with Beauveria bassiana on the 6th or 7th day, silkworms can spin cocoons, but the cocoons are softer and thinner than normal ones. When the cocoons are opened on the 4th day after cocooning, the silkworms have not yet pupated, their bodies have shortened, and they are completely dead. Therefore, it is not possible to produce dead pupae by inoculating large silkworms. (See Figure 1-Figure 4 )

[0048] 1.2 Inoculation during the pupal stage

[0049] 1.2.1 Live pupae

[0050] Take live silkworm pupae on the fourth day after cocooning, and inoculate them at a concentration of 5*10 7 After spraying the spore suspension of Beauveria bassiana, the mixture was cultured in an incubator at 26°C and 85% humidity until the mycelium showed no obvious growth. Figure 5 The medicinal material is plump, yellow-white in appearance, and the mycelium grows in the abdominal segment.

[0051] 1.2.2 Dead pupae

[0052] After killing silkworm pupae, sterilize them by high pressure, inoculate them and culture them in an incubator at 26℃ and 85% humidity until the mycelium has no obvious growth. Figure 6 ).

[0053] 2. Investigation of vaccination methods

[0054] Take live silkworm pupae on the fourth day after cocooning, and inoculate them at a concentration of 5*10 7 The cells were inoculated at 100 μg / mL using two inoculation methods: spraying (twice) and immersion (1 min). After inoculation, the cells were cultured in an incubator at 26°C and 85% humidity.

[0055] There is little difference in the appearance of the pupae formed by spraying and infiltration. Both show hyphae growing at the links, but the pupae are not fully covered with Beauveria bassiana. Moreover, the pupae gradually dehydrate and dry up after the fungus grows. The reason for this is that the pupae themselves do not take in nutrients after inoculation, so the Beauveria bassiana cannot get sufficient nutrients to grow.

[0056] The study found that the infiltration method can easily cause the silkworm pupae to turn black and fester. The reason is that the silkworm pupae are very fragile, and the longer the infiltration time, the greater the possibility of infection by miscellaneous bacteria. Therefore, in general, spray inoculation is better.

[0057] (See Figure 7-Figure 9 )

[0058] 3. Investigation of inoculation concentration

[0059] Spray inoculation was adopted, and the inoculation concentration was 5*10 6 , 5*10 7 , 5*10 8 After inoculation, the cells were cultured in an incubator at 26°C and 85% humidity.

[0060] The pupal yield, 50-grain weight, and beauvericin content at different inoculation concentrations are shown in Table 1. SPSS analysis was used, and the inoculation concentration was 5*10 6 and 5*10 7 The yield of medicinal materials produced by the production of pupae was significantly higher than that of 5*10 8 There was no significant difference in the weight of 50 grains of pupae medicinal materials in the three groups. However, when the inoculation concentration was 5*10 7 Therefore, considering the yield of pupae medicinal materials, the weight of 50 grains and the content of beauvericin, the inoculation concentration was 10 7 / ml is excellent (see Figure 10-12 ).

[0061] Table 1 Beauvericin content in pupae treated with different methods

[0062]

[0063] 4. Training time inspection

[0064] Take the live silkworm pupae on the fourth day after cocooning, inoculate them with a concentration of 10 7 After inoculation, the cells were cultured in an incubator at 26°C and 85% humidity for 7 to 10 days.

[0065] The experimental results showed that the pupae of the silkworms on the 7th and 8th day of culture were shrunken and the mycelia did not evenly cover the body surface; the pupae of the 10th day of culture were relatively full, but the mycelia only grew in the abdominal segment. It is speculated that this is because the silkworm pupae do not eat and cannot obtain nutrients from the outside. After the nutrients of the silkworms are exhausted by the Beauveria, the Beauveria cannot continue to grow (see Figure 13-16 ).

[0066] 5. Investigation of vaccination frequency

[0067] Take the live silkworm pupae on the fourth day after cocooning, inoculate them with a concentration of 10 7 / mL, spraying was performed 2, 3, and 4 times respectively, and cultured in an incubator at 26°C and 85% humidity for 10 days. The experimental results are shown in Table 2. Figure 17-Figure 19 ).

[0068] Table 2 Results of investigation on the number of vaccinations

[0069]

[0070] After 4 times of spray inoculation, there is a lot of residual bacterial liquid in the shallow dish. The silkworm pupae are in an overly humid environment for a long time, which causes the silkworm pupae to turn black and deteriorate. Therefore, in order to obtain medicinal materials with good appearance and internal quality, it is advisable to inoculate 3 times.

[0071] 6. Investigation of inoculation density

[0072] Take the live silkworm pupae on the fourth day after cocooning, inoculate them with a concentration of 10 7 pcs / mL, set 5000 pcs / mL 2 4000 pieces / m 2 3000 pieces / m 2 The density was determined and the inoculation was carried out by spraying for 3 times. After inoculation, the cells were cultured in an incubator at a temperature of 26°C and a humidity of 85% for 10 days.

[0073] The inoculation density showed significant differences. Considering the intrinsic quality of the pupae, 3000 / m was selected. 2 , the actual production is 4000 pieces / m 2 The appropriate vaccination rate is 3000 / m 2 ~4000 pieces / m 2 It is appropriate.

[0074] Table 3 Inoculation density investigation results

[0075]

[0076] 7 Pre-treatment inspection

[0077] Live pupae inoculated with Beauveria bassiana are susceptible to contamination by other bacteria, leading to blackening and deterioration. Therefore, to reduce this contamination, the experimental water was switched from mineral water to sterile water, which was then disinfected with 75% ethanol. The results, shown in Table 4, show that the use of sterile water did not affect the success rate of pupation recovery. However, after disinfection with ethanol, the pupation recovery rate was significantly improved, exceeding that of the other groups.

[0078] Table 4 The yield of stiff pupae after different treatment methods

[0079]

[0080]

[0081] 8 key breeding techniques

[0082] After the cocoons were broken and the pupae were taken out, they were disinfected with 75% ethanol and then 7 Spray inoculation was performed 3 times with a density of 3000 cells / m 2 ~4000 pieces / m 2It is best to place it in an environment with a temperature of 23-28℃ and a relative humidity of 75-85% and cultivate it for 10 days.

[0083] Table 5 The artificial breeding method of the medicinal pupae of the present invention is different from the prior art

[0084]

[0085]

[0086] The pupae medicinal material produced by the present invention grows white mycelia at the abdominal part, and the pupae are complete, yellow-white, hard, and solid in cross section. The pupae medicinal material prepared by method 2 is dark brown, with poor mycelial growth, and the surface mycelium is mainly yellow-brown, brittle and fragile. Method 3 uses silkworm pupa powder to prepare, and the pupae produced are yellow-white flakes and fragile. The pupae medicinal material prepared by method 4 has yellow-white mycelia on the surface, and is brittle and fragile. The appearance of the pupae medicinal material is shown in FIG. Figure 20-23 .

[0087] To alleviate the shortage of Bombyx batryticatus, pupae were initially developed as a substitute for Bombyx batryticatus. This suggests that pupae and Bombyx batryticatus have similar effects and share the same active ingredient. However, existing methods have used ammonium oxalate as an indicator for evaluating the intrinsic quality of medicinal materials. However, ammonium oxalate decomposes into ammonia and oxalic acid upon entering the human body, which can burden the liver and kidneys and is therefore unsuitable as a quality indicator for Bombyx batryticatus pupae.

[0088] Therefore, the present invention only selects beauvericin as a quality control indicator. The above pupae medicinal material powder is passed through the Pharmacopoeia No. 4 sieve and the beauvericin content is determined by HPLC. The chromatographic conditions are as follows: the mobile phase is acetonitrile-water solution (70:30), the flow rate is 1.0 mL·min-1, the column temperature is 30°C, and the wavelength is 215 nm. The spectrum shows that only the pupae produced by the present invention contain beauvericin. The beauvericin peak appears at a retention time of about 27 minutes, as shown in FIG. Figure 24 The reason for this is that beauvericin is produced by the mycelium of Beauveria bassiana when it is inoculated into living pupae. It is a secondary metabolite that can only be produced by acting on living bodies. In addition to the present invention, other methods use dead pupae and their powder, and few use only living pupae as inoculation objects.

Claims

1. A method for cultivating medicinal pupae, characterized in that: The method is prepared by inoculating the pupae of the silkworm Bombyx mori Linnaeus of the Bombydae family and culturing and breeding the dried pupae with Beauveria bassiana (Bals.) Vuillant, and comprises the following steps: a. Breaking the cocoon and taking out the pupa, and disinfecting it with 75% ethanol; the pupa is a living pupa; b. With a concentration of 1*10 7 -5*10 7 Spray inoculation 2-3 times, with an inoculation density of 3000 cells / m 2 ~4000 pieces / m 2 appropriate; c. Place it in an environment with a temperature of 23-28℃ and a relative humidity of 75-85% and cultivate it for 7-10 days.

2. The method for cultivating medicinal pupae according to claim 1, wherein: The inoculation concentration in step b is 5*10 7 / ml; the spray inoculation frequency was 3 times.

3. The method for cultivating medicinal pupae according to claim 1, wherein: The inoculation density is 3000 cells / m 2 .

4. The method for cultivating medicinal pupae according to claim 1, wherein: c The culture time is 10 days.

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