TRADITIONAL CHINESE MEDICINE COMPOUND FOR PREVENTING AND TREATING AFRICAN SWINE FEVER, AND ITS EFFICIENCY ENHANCEMENT PROCESS AND APPLICATION

Abstract

A traditional Chinese medicine compound for preventing and treating African swine fever, its efficiency enhancement process, and application are provided, which relate to the field of fermentation technologies of traditional Chinese medicine by probiotics. A formula with the most apparent preventive and therapeutic effect on ASFV is obtained by comparing various traditional Chinese medicine prescriptions and self-drafted formulas, and the formula is made from Codonopsis pilosula, Atractylodes macrocephala, Wolfiporia cocos, Auckiandialappa lappa Decne., Terminalia chebula, Aconitum carmichaeli, Myristica fragrans, Cynanchum otophyllum, Dioscorea polystachya and Ziziphus jujuba. Based on the formula, fermentation treatment of multi probiotics is performed, Bacillus subtilis, Saccharomyces cerevisiae, Bifidobacterium and Lactobacillus acidophilus are inoculated for fermenting, and a liquid fermented mixture is mixed to obtain fermentation preparation. The traditional Chinese medicine compound has a definite therapeutic effect, reasonable compatibility, a wide range of materials, and a low price, which makes it easy to promote and apply.

Claims

1. (canceled)

2. A preparation method of a traditional Chinese medicine compound for preventing and treating African swine fever, wherein the traditional Chinese medicine compound is made from the following raw materials in parts by weight: 10-20 parts of Codonopsis pilosula, 10-20 parts of Atractylodes macrocephala, 10-20 parts of Wolfiporia cocos, 3-9 parts of Auckiandialappa lappa Decne., 5-15 parts of Terminalia chebula, 3-9 parts of Aconitum carmichaeli, 5-15 parts of Myristica fragrans, 9-15 parts of Cynanchum otophyllum, 20-40 parts of Dioscorea polystachya and 2-5 parts of Ziziphus jujuba; wherein the preparation method comprises: step 1, weighing, according to the parts by weight, the Codonopsis pilosula, the Atractylodes macrocephala, the Wolfiporia cocos, the Auckiandialappa lappa Decne., the Terminalia chebula, the Aconitum carmichaeli, the Myristica fragrans, the Cynanchum otophyllum, the Dioscorea polystachya and the Ziziphus jujuba to obtain a first mixture, mixing and crushing the first mixture to obtain mixed powder; step 2, mixing the mixed powder obtained in step 1 with a fermentation medium, placing the fermentation medium mixed with the mixed powder into a primary fermenter, inoculating Bacillus subtilis and Saccharomyces cerevisiae in the primary fermenter to obtain a second mixture, stirring the second mixture continuously at 30-38 Celsius degrees ( C.) for 12-48 hours (h); wherein air is introduced into the primary fermenter containing the second mixture during the stirring for aerobic fermentation to obtain an intermediate product; and step 3, mixing the intermediate product with the fermentation medium, placing the fermentation medium mixed with the intermediate product into a secondary fermenter, adding anaerobic fermentation microorganisms Lactobacillus acidophilus and Bifidobacterium into the secondary fermenter to obtain a third mixture, placing the secondary fermenter containing the third mixture into a fermentation bag with a one-way exhaust valve for anaerobic fermentation at 28-42 C. for 1-5 days (d) to obtain a liquid finish product as the traditional Chinese medicine compound; and wherein an inoculation ratio of the Bacillus subtilis:the Saccharomyces cerevisiae:the Bifidobacterium:the Lactobacillus acidophilus is 2:1:1:1, and contents of the Bacillus subtilis, the Saccharomyces cerevisiae, the Bifidobacterium and the Lactobacillus acidophilus are greater than or equal to 1.010.sup.7 colony forming units per liter (CFU/L).

3. The preparation method as claimed in claim 2, wherein a formula of the fermentation medium in the primary fermenter and the secondary fermenter comprises: 10 grams (g) of refined peptone, 40 g of glucose, 5 g of sodium acetate, 2 g of ammonium citrate, 0.1 g of polysorbate 80, 0.58 g of magnesium sulfate, 0.28 g of manganese sulfate and 1000 milliliters (mL) of distilled water; a power of hydrogen (pH) of the fermentation medium is adjusted to 6-7 by using a 10% sodium hydroxide solution, and then the fermentation medium is autoclaved at 121 C. for 20 minutes (min).

4. The preparation method, as claimed in claim 3, wherein in step 2, an addition ratio of the mixed powder and the fermentation medium is 10-25 g: 1 liter (L).

5. The preparation method as claimed in claim 2, wherein a fermentation condition in the primary fermenter and the secondary fermenter is as follows: 0.01-0.2 kilopascals (kPa) of pressure, 15-35 revolutions per minute (r/min) of stirring speed, and 6.1-7.2 of pH value.

6-11. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1 illustrates a schematic diagram of a tissue culture infectious dose 50 (TCID.sub.50) test result of a traditional Chinese medicine compound against ASFV according to an embodiment of the disclosure.

[0036] FIG. 2 illustrates a schematic diagram of the effect of tetra-strain fermentation products on the activity of the ASFV according to an embodiment of the disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

[0037] Technical solutions in embodiments of the disclosure are clearly and completely described in conjunction with the embodiments of the disclosure below.

[0038] The embodiments of the disclosure are described in detail in conjunction with actual situations.

Embodiment 1 Identification of the Anti-ASFV Effect of a Traditional Chinese Medicine Compound

[0039] The traditional Chinese medicine compound of the disclosure is made from the following raw materials in parts by weight: 20 g of Codonopsis pilosula, 20 g of Atractylodes macrocephala, 20 g of Wolfiporia cocos, 9 g of Auckiandialappa lappa Decne., 15 g of Terminalia chebula, 9 g of Aconitum carmichaeli, 15 g of Myristica fragrans, 15 g of Cynanchum otophyllum, 40 g of Dioscorea polystachya and 5 g of Ziziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is fermented with Bacillus subtilis, Saccharomyces cerevisiae, Bifidobacterium and Lactobacillus acidophilus in stages to obtain the traditional Chinese medicine compound, and contents of the Bacillus subtilis, the Saccharomyces cerevisiae, the Bifidobacterium and the Lactobacillus acidophilus are greater than or equal to 1.010.sup.7 CFU/L. The virus strain used in the experiment is the ASFV gene type II strain (ASFV CN/SC/2019), which is an isolated strain from the African swine fever regional laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, a virus potency is 510.sup.7 TCID.sub.50/mL, a multiplicity of infection (MOI) of the ASFV CN/SC/2019 virus strain is 0.1.

[0040] 1. Kidney cells of African Chlorocebus sabaeus (Vero cells) are cultivated as follows: 5 mL of serum (purchased from Biological industries) and 500 microliters (L) of double antibody (i.e., penicillin and streptomycin) are added into each 45 mL of a Dulbecco's modified Eagle's medium (DMEM), and the DMEM added with the serum and the double antibody is stored in a refrigerator at 4 C. after mixing evenly.

[0041] 2. The Vero cells are divided into five groups that are a treatment group with a low dose of traditional Chinese medicine compound, a treatment group with a medium dose of traditional Chinese medicine compound (i.e., fermented liquid of Chinese medicine), a treatment group with a high dose of traditional Chinese medicine compound, a ribavirin group and a virus alone infection group (i.e., virus control), and each group has three replicates. In all groups, serum-free but 0.1% pancreatin-containing culture medium is used. The Vero cells with good growth status are inoculated at a 12-well plate, the serum-free but 0.1% pancreatin-containing culture medium is discarded when a cell confluence degree reaches 90%, and then the Vero cells are washed with phosphate-buffered saline (PBS) twice. The Vero cells are infected with the ASFV (MOI=0.1) in the treatment groups (i.e., the treatment group with the low dose of traditional Chinese medicine compound, the treatment group with the medium dose of traditional Chinese medicine compound, the treatment group with the high dose of traditional Chinese medicine compound) and the virus alone infection group, after 1 h of virus adsorption, the Vero cells are washed with PBS twice. The treatment groups are added with 1.5 mL of the traditional Chinese medicine compound (concentrations are 250 micrograms per milliliter abbreviated as g/mL, 500 g/mL and 1000 g/mL, respectively) and continuously cultivated for 24 h. Pathological conditions of the Vero cells are observed and photographed under a microscope.

[0042] 3. As samples, the 12-well plate with the Vero cells from step 2 is collected and placed at 80 C. for three freeze-thaw cycles. DMEM without serum is used to dilute the samples 10 times continuously to obtain samples with 10 dilutions; each dilution is repeated in 8 wells and inoculated until they are complete for culture, and the cell plates inoculated with the samples are cultured under 37 C. and 5% carbon dioxide (CO.sub.2) conditions. The pathological condition in each cell culture well is observed and recorded daily for 5 consecutive days. TCID.sub.50 of the virus solution is calculated according to a Reed-Muench formula. TCID.sub.50=logarithm of reciprocal of high critical dilution for 50% infection+distance ratiologarithm of dilution coefficient.

[0043] As shown in FIG. 1, the result shows that compared to the virus alone infection group, a virus titer (P<0.05) in the treatment group with low dose traditional Chinese medicine compound is significantly decreased, virus titers (P<0.01) in the treatment group with medium dose traditional Chinese medicine compound and the treatment group with high dose traditional Chinese medicine compound are extremely significantly decreased. Therefore, the traditional Chinese medicine compound's antiviral effect is dependent

Embodiment 2 Influence of Tetra-Strain Fermentation Product on the Activity of the ASFV

[0044] The tetra-strain fermentation product (i.e., traditional Chinese medicine compound fermented preparation) of the disclosure is made from the following raw materials in parts by weight: 10 g of Codonopsis pilosula, 10 g of Atractylodes macrocephala, 10 g of Wolfiporia cocos, 3 g of Auckiandialappa lappa Decne., 5 g of Terminalia chebula, 3 g of Aconitum carmichaeli, 5 g of Myristica fragrans, 9 g of Cynanchum otophyllum, 20 g of Dioscorea polystachya and 2 g of Ziziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and the Bacillus subtilis and the Saccharomyces cerevisiae are inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 37 C. for 24 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, and anaerobic fermentation microorganisms Lactobacillus acidophilus and Bifidobacterium are added into the secondary fermenter with half of the intermediate product. The secondary fermenter is placed into a fermentation bag with a one-way exhaust valve for anaerobic fermentation at 33 C. for 3 d to obtain a fermented mixture. Contents of the Bacillus subtilis, the Saccharomyces cerevisiae, the Bifidobacterium and the Lactobacillus acidophilus are greater than or equal to 1.010.sup.7 CFU/L. Fermentation effects of different combinations of the Bacillus subtilis, Saccharomyces cerevisiae, Bifidobacterium and Lactobacillus acidophilus are compared, and ratios of the different combinations are 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, respectively. The fermented mixture is directly aseptically filled in a fluid dosage form. A low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics. The virus strain used in the experiment is the ASFV gene type II strain (ASFV CN/SC/2019), which is an isolated strain from the African swine fever regional laboratory of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, a virus potency is 510.sup.7 TCID.sub.50/mL, MOI of the ASFV CN/SC/2019 virus strain is 0.1.

[0045] 1. Kidney cells of African Chlorocebus sabaeus (Vero cells) are cultivated as follows: 5 mL of serum (purchased from Biological industries) and 500 L of double antibody (i.e., penicillin and streptomycin) are added into each 45 mL of DMEM, and the DMEM added with the serum and the double antibody is stored in a refrigerator at 4 C. after mixing evenly.

[0046] 2. The Vero cells are divided into five groups that are fermentation groups with the different combinations of the four bacteria with ratios of 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, and a pure traditional Chinese medicine compound group, and each group has three replicates. In all groups, serum-free but 0.1% pancreatin-containing culture medium is used. The Vero cells with good growth status are inoculated at a 12-well plate, the serum-free but 0.1% pancreatin-containing culture medium is discarded when a cell confluence degree reaches 90%, and then the Vero cells are washed with PBS twice. The Vero cells are infected with the ASFV (MOI=0.1) in the fermentation groups and the pure traditional Chinese medicine compound group; after 1 h of virus adsorption, the Vero cells are washed with PBS twice. The fermentation and pure traditional Chinese medicine compound groups are added with 1.5 mL of the traditional Chinese medicine compound (concentrations are 500 g/mL). They are continuously cultivated for 24 hours to collect samples.

[0047] 3. DMEM without serum is used to dilute the samples 10 times continuously to obtain samples with 10 dilutions; each dilution is repeated in 8 wells and inoculated until they are complete for culture, and the cell plates inoculated with the samples are cultured under 37 C. and 5% CO.sub.2 conditions. The pathological condition in each cell culture well is observed and recorded daily for 5 consecutive days. TCID.sub.50 of the virus solution is calculated according to the Reed-Muench formula. TCID.sub.50=logarithm of reciprocal of high critical dilution for 50% infection+distance ratiologarithm of dilution coefficient.

[0048] As shown in FIG. 2, the fermentation effects of the different combinations of Bacillus subtilis, Saccharomyces cerevisiae, Bifidobacterium and Lactobacillus acidophilus are compared, the ratios of the different combinations are 1:1:1:1, 1:2:2:1, 1:1:2:2 and 2:1:1:1, respectively, and the different combinations are labelled as ASFV+1, ASFV+2, ASFV+3 and ASFV+4. It can be seen from the result that the antiviral effects of the fermentation groups are more significant than the pure traditional Chinese medicine compound group, and the ASFV+4 group has the optimal antiviral effect. That is, when the ratio of the combination is 2:1:1:1, the virus titer is the lowest, and the antiviral effect is optimal.

Embodiment 3 Actual Prevention Effect of the Tetra-Strain Fermentation Product on the ASFV

[0049] A traditional Chinese medicine compound fermented preparation for treating ASFV is provided. The traditional Chinese medicine compound fermented preparation is made from the following raw materials in parts by weight: 15 g of Codonopsis pilosula, 15 g of Atractylodes macrocephala, 15 g of Wolfiporia cocos, 6 g of Auckiandialappa lappa Decne., 10 g of Terminalia chebula, 6 g of Aconitum carmichaeli, 10 g of Myristica fragrans, 12 g of Cynanchum otophyllum, 30 g of Dioscorea polystachya and 3 g of Ziziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and the Bacillus subtilis and the Saccharomyces cerevisiae are inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 38 C. for 48 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, anaerobic fermentation microorganisms Lactobacillus acidophilus and Bifidobacterium are added into the secondary fermenter with half of the intermediate product, and the secondary fermenter is placed into a fermentation bag with one-way exhaust valve for anaerobic fermentation at 37 C. for 2 d to obtain a fermented mixture. Contents of the Bacillus subtilis, the Saccharomyces cerevisiae, the Bifidobacterium and the Lactobacillus acidophilus are greater than or equal to 1.010.sup.7 CFU/L.

[0050] The fermented mixture is directly aseptically filled in a fluid dosage form. A low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics.

[0051] A pig farm in Nanyang, Henan, has over 3000 sows. From April to June 2021, an ASF epidemic broke out in the surrounding pig farms of this pig farm, which once caused more than 90% of the pig farms to close down and clear the fields. The site selection of the pig farm has a reasonable layout, scientific pig farm management, and strict disinfection and prevention measures for people, cars and animals entering the farm. The pig farm controls biological safety well and focuses on protecting susceptible pig populations. The traditional Chinese medicine preparations were mixed into pig feed for feeding the pig based on the weight of freeze-dried traditional Chinese medicine compound once a day. The feeding ratio of the frozen-dried traditional Chinese medicine compound and the pig weights is 0.1 g: 1 kg, and the traditional Chinese medicine compound is used for a long time during the ASFV. At the beginning of this study, 600 sows were selected for experimental verification. The 600 sows were divided into 3 groups: the first group was blank, the second group was a traditional Chinese medicine compound fermented preparation control group, and the third group was a traditional Chinese medicine compound group, which lasted 7 days and was observed and recorded at any time.

TABLE-US-00001 TABLE 1 Prevention effect of traditional Chinese medicine compound and fermentation preparation thereof on sows in a pig farm Number Starting End Daily Death of testing Survival average average weight toll Group animals Mortality rate weight (kg) weight (kg) gain (g) (head) Blank group 200 1.5% 98.5% 80.8 87.5 956.67 3 Traditional Chinese 200 0.5% 99.5% 81.2 90.86 1380 1 medicine compound fermented preparation group Traditional Chinese 200 1% 99% 80.6 89.28 1240 2 medicine compound group

[0052] After 7 days of feeding, the daily weight gain of the traditional Chinese medicine compound fermented preparation group is higher than that of the traditional Chinese medicine compound group and the blank group, and the mortality rate of sows is reduced, the survival rate and the daily weight gain of sows are significantly improved.

Embodiment 4 Actual Treatment Effect of the Tetra-Strain Fermentation Product of the Traditional Chinese Medicine Compound on the ASFV

[0053] A traditional Chinese medicine compound fermented preparation for treating ASFV is provided. The traditional Chinese medicine compound fermented preparation is made from the following raw materials in parts by weight: 20 g of Codonopsis pilosula, 20 g of Atractylodes macrocephala, 20 g of Wolfiporia cocos, 9 g of Auckiandialappa lappa Decne., 15 g of Terminalia chebula, 9 g of Aconitum carmichaeli, 15 g of Myristica fragrans, 15 g of Cynanchum otophyllum, 40 g of Dioscorea polystachya and 5 g of Ziziphus jujuba. The raw materials are mixed and crushed to obtain mixed powder. The mixed powder is placed into a primary fermenter, and the Bacillus subtilis and the Saccharomyces cerevisiae are inoculated in the primary fermenter to obtain a mixture; the mixture is stirred continuously at 38 C. for 48 h. During stirring, air is introduced into the primary fermenter containing the mixture for aerobic fermentation to obtain an intermediate product. Half of the intermediate product is placed into a secondary fermenter, anaerobic fermentation microorganisms Lactobacillus acidophilus and Bifidobacterium are added into the secondary fermenter with half of the intermediate product, and the secondary fermenter is placed into a fermentation bag with one-way exhaust valve for anaerobic fermentation at 28 C. for 5 d to obtain a fermented mixture. Contents of the Bacillus subtilis, the Saccharomyces cerevisiae, the Bifidobacterium and the Lactobacillus acidophilus are greater than or equal to 1.010.sup.7 CFU/L. The fermented mixture is directly aseptically filled in a fluid dosage form, and low-temperature spray drying treatment is performed on the fermented mixture to obtain the traditional Chinese medicine preparation fermented with probiotics.

[0054] During the peak period of the ASFV from April to June 2021, the traditional Chinese medicine compound fermented preparation was used in a pig farm in Zhumadian, Henan Province, for pigs with symptoms. The traditional Chinese medicine compound fermented preparation was mixed with pig feed to feed the pig based on the weight of freeze-dried traditional Chinese medicine compound fermented preparation once a day. The feeding ratio of the frozen-dried traditional Chinese medicine compound and the pig weights is 0.2 g: 1 kg. During the ASFV, it was fed continuously for 7 days, and after symptom control, it was consolidated for 5 days. In the initial stage of this study, 300 live pigs were selected for the experiment and divided into three groups. The first group was a blank group, the second group was a traditional Chinese medicine compound fermented preparation control group, and the third was a traditional Chinese medicine compound group. Observation and recording were conducted at any time.

TABLE-US-00002 TABLE 2 Treatment effect of traditional Chinese medicine compound and fermentation preparation on sows in pig farm Number of Survival Death toll Group testing animals Mortality rate (head) Blank group 100 98 2% 98 Traditional Chinese 100 48 52% 48 medicine compound fermented preparation group Traditional Chinese 100 62 38% 62 medicine compound group

[0055] After 12 days of treatment, the traditional Chinese medicine compound fermented reparation group significantly reduced the mortality rate of live pigs and improved their survival rate.

[0056] It should be noted that the above-mentioned implementation schemes should be understood as explanatory and not limiting a scope of protection of the disclosure. The scope of protection of the disclosure shall be subject to claims. For those skilled in the art, some non-essential improvements and adjustments made to the disclosure, without departing from its essence and scope of the disclosure, still fall within the scope of protection of the disclosure.