CN112136972A - Method for producing feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines - Google Patents
Method for producing feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines Download PDFInfo
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Abstract
The invention discloses a method for producing a feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines, wherein antibiotics are the main means and method for preventing and treating bacterial infection of poultry, but are easy to remain in animal-derived food, so that the health of human beings is seriously harmed. Dandelion, rehmannia root and dyers woad leaf in cordyceps militaris and traditional Chinese medicines have bacteriostatic active ingredients, antibacterial resources are searched from traditional Chinese medicines and the like, and the screening of antibacterial medicines is becoming a new trend more and more. The invention utilizes the cordyceps militaris and the extract of the traditional Chinese medicines of dandelion, rehmannia root and dyers woad leaf after mixed fermentation to be added into the feed, and has important significance in the aspects of reducing the breeding cost, preventing the abuse of antibiotics in the breeding process of livestock, enhancing the food safety and the like.
Description
Technical Field
The invention relates to a method for producing a feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines.
Background
Cordyceps is a short name of cordyceps militaris, also called cordyceps militaris or cordyceps militaris, and is a medicinal fungus combining with insect fungi. Cordyceps in Clavipitaceae is one of the famous and precious Chinese medicinal materials. Modern pharmaceutical research shows that the cordyceps militaris contains nucleosides, polysaccharides, cordycepin, sterols and other components with the content basically the same as that of the cordyceps militaris, has a treatment effect on diseases of immune system, nervous system, cardiovascular system and the like, and has anti-tumor and anti-cancer effects.
The dandelion is a traditional Chinese medicine in China, has wide distribution range and strong vitality, and can be used for medicine. Clear heat and remove toxicity, induce diuresis and dissipate nodulation. It can be used for treating acute mastitis, lymphadenitis, lymphoid tuberculosis, furunculosis, acute conjunctivitis, common cold with fever, acute tonsillitis, acute bronchitis, gastritis, hepatitis, cholecystitis, and urinary tract infection.
Rehmanniae radix is fresh or dried root tuber of Scrophulariaceae, or dried rehmanniae radix, and can be used for treating fever with yin impairment, crimson tongue with dipsosis, epidemic heat syndrome, hematemesis, epistaxis, and sore throat.
Dyers woad leaf, name of traditional Chinese medicine. Macula due to toxic heat, erysipelas, sore throat, aphtha of the mouth and tongue, skin ulcer, carbuncle, and toxic swelling. In recent years, the medicine is widely applied clinically, and can be used for treating the symptoms and also can be used for treating phlegm heat stagnation in lung and yellow and thick expectoration; especially for epidemic encephalitis B, it can be used alone for prevention, and can be combined with chai Hu, Yin Hua, Lian Qiao, ban lan Gen, Xuan Shen, Sheng Di Huang, etc. to clear heat-toxicity in qi system and nutrient system, so it is suitable for various types of encephalitis B, especially for heat type. The folium isatidis sold in different places of China is of various varieties, and the plant sources are different: acanthaceae plant herba Kalimeridis. Isatis tinctoria and radix Isatidis belonging to Brassicaceae. Polygonum tinctorium of Polygonaceae. The plant of Leguminosae is Indigofera tinctoria. The leaves of the above plants are all used as dyers woad leaf, and can also be used as raw materials for preparing indigo naturalis, except for Indigowoad root, the roots of which are all used as isatis roots.
Compared with the traditional technical mode, the combined fermentation product of cordyceps militaris, dandelion and other traditional Chinese medicines has the characteristics of rapidness, accuracy, scale and the like, and the fermentation product has the characteristics of wide antibacterial spectrum, strong sterilization and bacteriostasis capability, certain antiviral activity and other biological characteristics.
In order to improve the antibacterial and disease-resistant capability of livestock, antibiotics are generally selected for the existing livestock feed additives and the prevention and treatment of diseases, and the main defects are as follows: firstly, the antibiotic residue in the livestock product is increased, and the livestock product has certain harm to the health of people after being eaten; after the antibiotics are excessively used, pathogenic bacteria generate drug resistance to the antibiotics, so that the immunity of the livestock is reduced, and the prevention and the treatment of the diseases of the livestock in the future are more and more difficult.
However, the traditional Chinese medicine contains a plurality of bacteriostatic active ingredients with strong activity and low toxicity, so that the people are concerned and supported more and more about finding antibacterial resources from natural plants such as the traditional Chinese medicine and screening antibacterial drugs.
The invention adopts cordycepin extracted from cordyceps militaris to be mixed with enzyme extract of traditional Chinese medicines such as dandelion, rehmannia root, isatis root and the like, and the raw medicinal liquid is formed by inactivation, filtration and concentration, thus enhancing the biological activities of the fermentation product such as bacteriostatic activity, antivirus, anti-inflammation, immunity improvement and the like.
At present, no report about the production of the antibiotic-free feed additive by the combined fermentation of the cordyceps militaris and the dandelion is provided at home and abroad.
Disclosure of Invention
The invention discloses a method for producing a feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines, wherein antibiotics are the main means and method for preventing and treating bacterial infection of poultry, but are easy to remain in animal-derived food, so that the health of human beings is seriously harmed. Dandelion, rehmannia root and dyers woad leaf in cordyceps militaris and traditional Chinese medicines have bacteriostatic active ingredients, antibacterial resources are searched from traditional Chinese medicines and the like, and the screening of antibacterial medicines is becoming a new trend more and more. The invention utilizes the cordyceps militaris and the extract of the traditional Chinese medicines of dandelion, rehmannia root and dyers woad leaf after mixed fermentation to be added into the feed, and has important significance in the aspects of reducing the breeding cost, preventing the abuse of antibiotics in the breeding process of livestock, enhancing the food safety and the like.
In order to solve the technical problems, the invention adopts the following technical scheme.
(with claims)
The invention is characterized in that:
the invention aims to overcome the defects of the prior art and provide the antibiotic-free livestock feed additive which has low production cost and strong biological activities such as bacteriostatic activity, antivirus activity, anti-inflammation activity, immunity improvement and the like, and replaces the production method of the existing antibiotic feed additive.
The object of the invention can be achieved by the following measures: the method for producing the antibiotic-free feed additive by combined fermentation of the cordyceps militaris, the dandelion, the rehmannia root, the isatis root and the like is characterized by short production period, solving drug resistance and mutation types and improving the biological activity of a fermentation product. The specific process steps are as follows:
(1) preparing a cordyceps culture medium:
the formula comprises the following components in parts by weight: 95.0% of potato liquid, 3% of traditional Chinese medicine liquid, 1% of glucose and 1% of peptone. About 250ml of water was added to each bottle, 20ml of water was added.
(2) Sterilizing a cordyceps culture medium:
autoclaving with autoclave, steam autoclaving at 121 deg.C under 0.1MP for 30 min
(3) Cooling and inoculating:
cooling in the cooling chamber to reduce the temperature of the high-temperature cultivation bottle to about 25 ℃. Inoculating the strain under aseptic condition.
(4) Fermentation culture:
culturing Cordyceps liquid culture medium in shaking table at 25 deg.C and rotation speed of 160 rpm for 7 days, and ending fermentation when mycelium pellet size is 0.1-0.2cm and mycelium pellet amount is 80%.
(5) Inactivating
The fermentation broth was transferred to a constant temperature inactivation tank and inactivated at 80 ℃ for 2 hours.
(6) Filtering and concentrating:
separating the mycelium pellets from the liquid by using a 200-mesh filter screen, and then carrying out the following steps: 10 to form a raw liquid medicine.
Drawings
FIG. 1: bacteriostatic effect diagram of traditional Chinese medicine extract stock solution
FIG. 2: bacteriostatic effect diagram of Chinese medicinal extractive solutions with different concentrations on Escherichia coli
FIG. 3: bacteriostatic effect diagram of traditional Chinese medicine extracting solutions with different concentrations on bacillus subtilis
FIG. 4: bacteriostatic effect diagram of traditional Chinese medicine extracting solutions with different concentrations on staphylococcus aureus
The specific implementation mode is as follows:
the following detailed description of the embodiments of the invention:
example 1:
preparing strains:
the Cordyceps militaris strain is obtained by separating and purifying wild Cordyceps militaris in Changbai mountain.
(1) Test tube slant strain culture (activation):
preparing a culture medium: cutting 200g of carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, taking juice, adding 20g of glucose, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and 20g of agar, melting, fixing the volume to 1000mL, and naturally adjusting the pH value; subpackaging in test tubes; inoculating the low-temperature preserved strain into the test tube slant under aseptic condition, and performing dark culture at 25 ℃ for 12 days.
(2) Preparation of liquid Medium
Cutting 200g carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, collecting juice, adding 20g glucose, 0.5g magnesium sulfate and 1g potassium dihydrogen phosphate, fixing volume to 1000mL, and naturally adjusting pH. Subpackaging in 500mL triangular flasks and keeping the pH value natural.
(II) sterilizing a culture medium:
the prepared culture medium is sterilized at 121 ℃ for 20 min.
(III) cooling and inoculating:
naturally cooling to 25 deg.C after sterilization, inoculating Cordyceps militaris activated strain under aseptic condition.
(IV) culturing:
culturing the inoculated Cordyceps militaris liquid culture medium in a shaking table at 25 deg.C and 160 rpm for 5 days.
(V) selecting:
the mycelium pellet size is 1-2mm, and the fermentation liquid is clear and transparent and has no layering phenomenon. As a liquid seed culture.
Preparing traditional Chinese medicine enzyme extracting solution:
putting dandelion, isatis root and rehmannia root into an enzymolysis tank according to the ratio of 3:4:3, and adding distilled water to obtain a liquid medicine mixture, wherein the weight ratio of the traditional Chinese medicines to the distilled water is 1: 10, then adding cellulose degrading enzyme and protein degrading enzyme, wherein the adding amount of the cellulose degrading enzyme and the protein degrading enzyme is 1-1.5 percent and 0.5-1 percent of the total weight of the liquid medicine mixture respectively, degrading for 24 hours at the constant temperature of 50 ℃, and finally taking out and filtering to obtain filtrate, namely the traditional Chinese medicine enzyme extracting solution.
(VII) preparation of a mixed fermentation medium of cordyceps militaris and traditional Chinese medicines:
the formula comprises the following components in parts by weight: 95.0% of potato liquid, 3% of traditional Chinese medicine liquid, 1% of glucose and 1% of peptone. About 250ml of water was added to each bottle, 20ml of water was added.
(2) Sterilizing a cordyceps culture medium:
autoclaving with autoclave, steam autoclaving at 121 deg.C under 0.1MP for 30 min
(3) Cooling and inoculating:
cooling in the cooling chamber to reduce the temperature of the high-temperature cultivation bottle to about 25 ℃. Inoculating the strain under aseptic condition.
(4) Fermentation culture:
culturing Cordyceps liquid culture medium in shaking table at 25 deg.C and rotation speed of 160 rpm for 7 days, and ending fermentation when mycelium pellet size is 0.1-0.2cm and mycelium pellet amount is 80%.
(6) Inactivating
The fermentation broth was transferred to a constant temperature inactivation tank and inactivated at 80 ℃ for 2 hours.
(7) Filtering and concentrating:
separating the mycelium pellets from the liquid by using a 200-mesh filter screen, and then carrying out the following steps: 10 to form a raw liquid medicine.
Example 2:
preparing strains:
the Cordyceps militaris strain is obtained by separating and purifying wild Cordyceps militaris in Changbai mountain.
(1) Test tube slant strain culture (activation):
preparing a culture medium: cutting 200g of carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, taking juice, adding 20g of glucose, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and 20g of agar, melting, fixing the volume to 1000mL, and naturally adjusting the pH value; subpackaging in test tubes; inoculating the low-temperature preserved strain into the test tube slant under aseptic condition, and performing dark culture at 25 ℃ for 12 days.
(2) Preparation of liquid Medium
Cutting 200g carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, collecting juice, adding 20g glucose, 0.5g magnesium sulfate and 1g potassium dihydrogen phosphate, fixing volume to 1000mL, and naturally adjusting pH. Subpackaging in 500mL triangular flasks and keeping the pH value natural.
(II) sterilizing a culture medium:
the prepared culture medium is sterilized at 121 ℃ for 20 min.
(III) cooling and inoculating:
naturally cooling to 25 deg.C after sterilization, inoculating Cordyceps militaris activated strain under aseptic condition.
(IV) culturing:
culturing the inoculated Cordyceps militaris liquid culture medium in a shaking table at 25 deg.C and 160 rpm for 5 days.
(V) selecting:
the mycelium pellet size is 1-2mm, and the fermentation liquid is clear and transparent and has no layering phenomenon. As a liquid seed culture.
Preparing traditional Chinese medicine enzyme extracting solution:
putting dandelion, isatis root and rehmannia root into an enzymolysis tank according to the ratio of 3:4:3, and adding distilled water to obtain a liquid medicine mixture, wherein the weight ratio of the traditional Chinese medicines to the distilled water is 1: 10, then adding cellulose degrading enzyme and protein degrading enzyme, wherein the adding amount of the cellulose degrading enzyme and the protein degrading enzyme is 1-1.5 percent and 0.5-1 percent of the total weight of the liquid medicine mixture respectively, degrading for 24 hours at the constant temperature of 50 ℃, and finally taking out and filtering to obtain filtrate, namely the traditional Chinese medicine enzyme extracting solution.
(VIII) preparing a cordyceps militaris and traditional Chinese medicine mixed fermentation medium:
the formula comprises the following components in parts by weight: 95.0% of potato liquid, 4% of traditional Chinese medicine liquid, 1% of glucose and 1% of peptone. About 250ml of water was added to each bottle, 20ml of water was added.
(1) Sterilizing a cordyceps culture medium:
autoclaving with autoclave, steam autoclaving at 121 deg.C under 0.1MP for 30 min
(2) Cooling and inoculating:
cooling in the cooling chamber to reduce the temperature of the high-temperature cultivation bottle to about 25 ℃. Inoculating the strain under aseptic condition.
(3) Fermentation culture:
culturing Cordyceps liquid culture medium in shaking table at 25 deg.C and rotation speed of 160 rpm for 7 days, and ending fermentation when mycelium pellet size is 0.1-0.2cm and mycelium pellet amount is 80%.
(4) Inactivating
The fermentation broth was transferred to a constant temperature inactivation tank and inactivated at 80 ℃ for 2 hours.
(5) Filtering and concentrating:
separating the mycelium pellets from the liquid by using a 200-mesh filter screen, and then carrying out the following steps: 10 to form a raw liquid medicine.
Example 3:
preparing strains:
the Cordyceps militaris strain is obtained by separating and purifying wild Cordyceps militaris in Changbai mountain.
(1) Test tube slant strain culture (activation):
preparing a culture medium: cutting 200g of carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, taking juice, adding 20g of glucose, 0.5g of magnesium sulfate, 1g of monopotassium phosphate and 20g of agar, melting, fixing the volume to 1000mL, and naturally adjusting the pH value; subpackaging in test tubes; inoculating the low-temperature preserved strain into the test tube slant under aseptic condition, and performing dark culture at 25 ℃ for 12 days.
(2) Preparation of liquid Medium
Cutting 200g carrot into small blocks of 0.8-1.2cm (optimally 1 cm), boiling in boiling water for 20 minutes, filtering, collecting juice, adding 20g glucose, 0.5g magnesium sulfate and 1g potassium dihydrogen phosphate, fixing volume to 1000mL, and naturally adjusting pH. Subpackaging in 500mL triangular flasks and keeping the pH value natural.
(II) sterilizing a culture medium:
the prepared culture medium is sterilized at 121 ℃ for 20 min.
(III) cooling and inoculating:
naturally cooling to 25 deg.C after sterilization, inoculating Cordyceps militaris activated strain under aseptic condition.
(IV) culturing:
culturing the inoculated Cordyceps militaris liquid culture medium in a shaking table at 25 deg.C and 160 rpm for 5 days.
(V) selecting:
the mycelium pellet size is 1-2mm, and the fermentation liquid is clear and transparent and has no layering phenomenon. As a liquid seed culture.
Preparing traditional Chinese medicine enzyme extracting solution:
putting dandelion, isatis root and rehmannia root into an enzymolysis tank according to the ratio of 3:4:3, and adding distilled water to obtain a liquid medicine mixture, wherein the weight ratio of the traditional Chinese medicines to the distilled water is 1: 10, then adding cellulose degrading enzyme and protein degrading enzyme, wherein the adding amount of the cellulose degrading enzyme and the protein degrading enzyme is 1-1.5 percent and 0.5-1 percent of the total weight of the liquid medicine mixture respectively, degrading for 24 hours at the constant temperature of 50 ℃, and finally taking out and filtering to obtain filtrate, namely the traditional Chinese medicine enzyme extracting solution.
(VII) preparation of a mixed fermentation medium of cordyceps militaris and traditional Chinese medicines:
the formula comprises the following components in parts by weight: 95.0% of potato liquid, 5% of traditional Chinese medicine liquid, 1% of glucose and 1% of peptone. About 250ml of water was added to each bottle, 20ml of water was added.
(2) Sterilizing a cordyceps culture medium:
autoclaving with autoclave, steam autoclaving at 121 deg.C under 0.1MP for 30 min
(3) Cooling and inoculating:
cooling in the cooling chamber to reduce the temperature of the high-temperature cultivation bottle to about 25 ℃. Inoculating the strain under aseptic condition.
(4) Fermentation culture:
culturing Cordyceps liquid culture medium in shaking table at 25 deg.C and rotation speed of 160 rpm for 7 days, and ending fermentation when mycelium pellet size is 0.1-0.2cm and mycelium pellet amount is 80%.
(5) Inactivating
The fermentation broth was transferred to a constant temperature inactivation tank and inactivated at 80 ℃ for 2 hours.
(6) Filtering and concentrating:
separating the mycelium pellets from the liquid by using a 200-mesh filter screen, and then carrying out the following steps: 10 to form a raw liquid medicine.
During the period from 10 months in 2018 to 5 months in 2019, the present inventors and their laboratory staff performed the following experiments.
An oxford cup method is adopted to respectively carry out bacteriostatic experiments on three bacteria, namely staphylococcus chromogenes, escherichia coli and bacillus subtilis, and the experimental results are as follows:
the observation of the inhibition zone is carried out by adopting an oxford cup method, and the shape and the size of the inhibition zone in experimental results of various methods are compared through observation. The results recorded when the oxford cup was subjected to the experiment are shown in figure 1.
Through analysis and comparison of experimental data, the following conclusions are drawn:
1. the traditional Chinese medicine extract has good bacteriostatic effect on escherichia coli, bacillus subtilis and staphylococcus aureus in vitro. And the bacteriostatic effect of the traditional Chinese medicine extract consisting of the mulberry leaf extract, the folium artemisiae argyi extract and the purslane extract on escherichia coli, staphylococcus aureus and bacillus subtilis is gradually weakened along with the increase of the dilution times of the traditional Chinese medicine extract.
2. The highest dilution times of the traditional Chinese medicine extracting solution with the bacteriostatic effect are about 100 times, and the bacteriostatic effect of the traditional Chinese medicine extracting solution diluted by 100 times on escherichia coli, bacillus subtilis and staphylococcus aureus is not obvious.
3. The Escherichia coli is least sensitive to the reaction of antibiotics and traditional Chinese medicine extract, the bacteriostatic effect of the traditional Chinese medicine extract stock solution on the bacillus subtilis and the staphylococcus aureus is better than that of the Escherichia coli in terms of the diameter of a bacteriostatic circle, the diameter of the bacteriostatic circle is not up to 4cm at most, but is about 2cm and far lower than that of the staphylococcus aureus and the bacillus subtilis.
The experiment verifies that the traditional Chinese medicine extracting solution has good in-vitro antibacterial effect and has important significance for reducing the use of antibiotics in future breeding and food safety treatment.
Claims (4)
1. A method for producing a feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines is characterized by comprising the following steps: adding enzyme extract of traditional Chinese medicines such as dandelion, rehmannia root and folium isatidis in the cordyceps militaris liquid fermentation process, culturing a cordyceps militaris liquid culture medium in a shaking table at 25 ℃ and 160 revolutions for 7 days, transferring the cordyceps militaris liquid culture medium into a constant-temperature inactivation tank when the mycelium pellet size is 0.1-0.2cm and the mycelium pellet amount is 80%, inactivating the cordyceps militaris for 2 hours at 80 ℃, and filtering the cordyceps militaris at 200 meshes to obtain the cordyceps militaris and traditional Chinese medicine combined fermentation production feed additive.
2. The method for producing the feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines according to claim 1, wherein the method for extracting the traditional Chinese medicine enzymes in the enzyme extracting solutions of the traditional Chinese medicines of dandelion, rehmannia root and folium isatidis comprises the steps of putting the dandelion, the radix isatidis and the rehmannia root into an enzymolysis tank according to the ratio of 3:4:3, adding distilled water to obtain a liquid medicine mixture, wherein the weight ratio of the traditional Chinese medicines to the distilled water is 1: 10, then adding cellulose degrading enzyme and protein degrading enzyme, wherein the adding amount of the cellulose degrading enzyme and the protein degrading enzyme is 1-1.5 percent and 0.5-1 percent of the total weight of the liquid medicine mixture respectively, degrading for 24 hours at the constant temperature of 50 ℃, and finally taking out and filtering to obtain filtrate, namely the traditional Chinese medicine enzyme extracting solution.
3. The method for producing the feed additive by combined fermentation of cordyceps militaris and traditional Chinese medicines according to claim 1 or 2, which is characterized by comprising the following steps of:
the invention aims to overcome the defects of the prior art and provide the antibiotic-free livestock feed additive which has low production cost and strong biological activities such as bacteriostatic activity, antivirus activity, anti-inflammation activity, immunity improvement and the like, and replaces the production method of the existing antibiotic feed additive.
4. The object of the invention can be achieved by the following measures: the method for producing the antibiotic-free feed additive by combined fermentation of the cordyceps militaris, the dandelion, the rehmannia root, the isatis root and the like is characterized by short production period, solving drug resistance and mutation types and improving the biological activity of a fermentation product.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091100A (en) * | 2009-12-15 | 2011-06-15 | 云南云百草生物技术有限公司 | Method for improving main medicinal ingredients through solid fermentation of Indian buead and cordyceps militaris |
| CN108812051A (en) * | 2018-05-23 | 2018-11-16 | 鲁东大学 | A kind of cordyceps culturing method |
| CN108901615A (en) * | 2018-07-19 | 2018-11-30 | 保定学院 | Utilize the method for trollflower pharmacological property culture medium culture cordyceps mycelium |
| CN109010382A (en) * | 2018-09-08 | 2018-12-18 | 河南欧普生物科技有限公司 | A kind of preparation method of fermentation cordyceps oral solution for animals |
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- 2019-06-28 CN CN201910570862.0A patent/CN112136972A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102091100A (en) * | 2009-12-15 | 2011-06-15 | 云南云百草生物技术有限公司 | Method for improving main medicinal ingredients through solid fermentation of Indian buead and cordyceps militaris |
| CN108812051A (en) * | 2018-05-23 | 2018-11-16 | 鲁东大学 | A kind of cordyceps culturing method |
| CN108901615A (en) * | 2018-07-19 | 2018-11-30 | 保定学院 | Utilize the method for trollflower pharmacological property culture medium culture cordyceps mycelium |
| CN109010382A (en) * | 2018-09-08 | 2018-12-18 | 河南欧普生物科技有限公司 | A kind of preparation method of fermentation cordyceps oral solution for animals |
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Application publication date: 20201229 |