CN112136965B - Immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive and preparation method thereof - Google Patents
Immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive and preparation method thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/105—Aliphatic or alicyclic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/26—Compounds containing phosphorus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Biotechnology (AREA)
- Birds (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Mycology (AREA)
- Botany (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention aims to provide a propagation-free growth-promoting fermented Chinese herbal medicine feed additive, which is used for livestock and poultry and comprises a Chinese herbal medicine mixture and probiotics for fermenting the Chinese herbal medicine mixture, wherein the probiotics are used for fermenting the Chinese herbal medicine mixture to prepare a final product; the Chinese herbal medicine mixture comprises: 9-15 parts of folium artemisiae argyi, 6-12 parts of pongamia pinnata, 9-18 parts of hovenia dulcis, 5-15 parts of seaweed and 5-18 parts of elephant grass; the probiotics is at least one of bacillus subtilis, bacillus licheniformis, lactobacillus plantarum and saccharomyces cerevisiae. The feed additive has immunity enhancing and growth promoting effects, and has effects of improving drug effect, producing synergistic effect, and reducing toxic and side effects of Chinese herbal medicine. The preparation method is simple, can increase the utilization rate of the effective components of the Chinese herbal medicine, reduce the adverse reaction of the raw materials of the Chinese herbal medicine and improve the palatability of the Chinese herbal medicine.
Description
Technical Field
The invention belongs to the field of feed additive formula design and traditional Chinese medicine fermentation, and particularly relates to a fermented Chinese herbal medicine feed additive capable of enhancing immunity and promoting growth and a preparation method thereof.
Background
The growth-promoting feed additive is used in large quantities in daily livestock and poultry production, and has increasingly obvious defects while bringing great promotion effect on animal production. Therefore, the official policy of the rural part of agriculture starts from 7/1/2020, feed production enterprises stop producing commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicines), and clearly indicate that all growth-promoting drug feed additive varieties except traditional Chinese medicines cannot be produced and imported. In this context, the traditional Chinese medicine growth-promoting feed additive will be supported and developed.
Chinese herbal medicine resources in China are very rich, play an important role in livestock and poultry health care, can promote animal growth, improve the quality of livestock and poultry products and enhance the immunity of animal organisms, has the characteristics of low toxicity and low residue, is a nutrient substance, can be used as a beneficial supplement for nutrient components in feed, and meets the requirement of the animal organisms on balanced nutrition. However, the existing Chinese herbal medicine feed additive is too simple to use and has the problems of large dosage, slow effect, poor palatability, toxic and side effect and the like.
The biological fermentation technology is utilized to release a large amount of effective active ingredients in the Chinese herbal medicines rapidly under mild reaction conditions, and simultaneously, new active ingredients are generated, and the toxic and side effects of the Chinese herbal medicines are reduced. The double effects of promoting the growth of animals and enhancing the immunity of the animal organism in the production of animals by the probiotics and the Chinese herbal medicines are exerted.
Disclosure of Invention
The invention aims to provide a immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive, the Chinese herbal medicines used in the feed additive have the effects of enhancing immunity and promoting growth, the efficacy of the feed additive is improved through a probiotic fermentation technology, a synergistic effect is generated, and the toxic and side effects of the Chinese herbal medicines are reduced.
The second purpose of the invention is to provide a preparation method of the feed additive, which is simple, can increase the utilization rate of the effective components of the Chinese herbal medicines, reduce the adverse reaction of the raw materials of the Chinese herbal medicines and improve the palatability of the Chinese herbal medicines.
The object of the invention is achieved in the following way: a fermented Chinese herbal medicine feed additive for livestock and poultry with immunity enhancing and growth promoting effects comprises Chinese herbal medicine mixture and probiotic bacteria for fermenting the Chinese herbal medicine mixture, and the probiotic bacteria are used for fermenting the Chinese herbal medicine mixture to obtain final product;
the Chinese herbal medicine mixture comprises: 9-15 parts of folium artemisiae argyi, 6-12 parts of pongamia pinnata, 9-18 parts of hovenia dulcis, 5-15 parts of seaweed and 5-18 parts of elephant grass;
the probiotics is at least one of bacillus subtilis, bacillus licheniformis, lactobacillus plantarum and saccharomyces cerevisiae.
The probiotics comprise 40 parts by weight of bacillus subtilis and 60 parts by weight of bacillus licheniformis.
The preparation method of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock and poultry comprises the steps of preparing the Chinese herbal medicine mixture into a Chinese herbal medicine fermentation culture medium, inoculating seed liquid of probiotics into the Chinese herbal medicine fermentation culture medium, and fermenting.
The Chinese herbal medicine fermentation medium is obtained by the following steps:
pulverizing the Chinese herbal medicines, mixing uniformly, adding 100-500 parts by weight of water, boiling for 1-3h, then adding glucose, urea, dipotassium hydrogen phosphate, magnesium sulfate and sodium chloride, and mixing uniformly.
The Chinese herbal medicine mixture, the glucose, the urea, the dipotassium hydrogen phosphate, the magnesium sulfate and the sodium chloride are sequentially mixed according to the weight ratio of 88-98:2-4:1-2:0.1-0.3:0.01-0.03: 0.3-0.9.
The seed liquid of the probiotics is obtained by the following steps:
respectively inoculating the preserved bacillus subtilis and bacillus licheniformis into a nutrient broth solid culture medium to activate for 22-26h, selecting single colonies with good growth, respectively inoculating into the nutrient broth solid culture medium, then culturing for 22-26h at 28-37 ℃, and mixing to obtain a bacterial suspension of the bacillus subtilis and the bacillus licheniformis; the components of the nutrient broth medium comprise peptone, beef extract and sodium chloride.
Respectively inoculating bacillus subtilis and bacillus licheniformis which are preserved by glycerol into a nutrient broth solid culture medium for activation; according to the weight ratio of 4:6 mixing the prepared bacterial suspension of the bacillus subtilis and the bacillus licheniformis; culturing at 28-37 deg.C for 22-26h at rotation speed of 180-220 r/min.
The mass of the inoculated seed liquid of the probiotics accounts for 3-7% of the mass of the Chinese herbal medicine fermentation medium.
The fermentation is carried out for 56-72h at the temperature of 32-36 ℃; after fermentation, drying the fermentation product until the water content is not more than 10wt%; drying, pulverizing the dried product, and sieving with 80-100 mesh sieve.
The fermentation is carried out for 64h at 35 ℃.
The invention has the beneficial effects that:
the invention provides a livestock and poultry immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive, which comprises a Chinese herbal medicine mixture and probiotics for fermenting the Chinese herbal medicine mixture, wherein the probiotics are used for fermenting the Chinese herbal medicine mixture to prepare a final product; the Chinese herbal mixture comprises folium Artemisiae Argyi, cortex Phellodendri, semen Hoveniae, sargassum and herba elephantopi.
(1) The antibacterial activity of the phellodendron amurense rupr and the folium artemisiae argyi are enhanced through compatibility, and the capability of resisting diseases is enhanced; the hovenia dulcis thunb and the elephant grass are matched, so that the crude fiber, the crude protein, the crude fat, the total sugar, the vitamin and other nutrient substances are rich, the proportion is proper, and the weight gain is obvious. The fermented Chinese herbal medicine feed additive for enhancing immunity and promoting growth for livestock and poultry provided by the embodiment of the invention has good palatability, can obviously enhance the feed intake of livestock and poultry, and has an obvious weight increasing effect. The method is proved in breeding experiments of chicken, duck and pig.
(2) Various enzymes can be secreted by probiotic fermentation, and a large amount of effective components in the cell walls of the Chinese herbal medicines are dissociated to facilitate the absorption of organisms, so that the drug effect is enhanced, the dosage is reduced, and the problem of slow response of the Chinese herbal medicines is solved. Especially the combination of bacillus subtilis and bacillus licheniformis.
(3) Reduce toxic and side effects and increase the acceptance of animals. The Chinese herbal medicines used in the embodiment of the invention, such as wampee, seaweed and the like, have certain toxic and side effects if not processed, are bitter in taste, can seriously reduce the feed intake of animals when being added into feed, and the Chinese herbal medicines subjected to fermentation treatment greatly reduce the bitter taste on the premise of ensuring the drug effect, have almost no toxic and side effects, and have the possibility of generating new drug-effect substances.
(4) The probiotics and the Chinese herbal medicines have synergistic effect. The fermentation strains of the bacillus subtilis and the bacillus licheniformis used in the embodiment of the invention are probiotics and have the functions of promoting the growth and development of animals and improving the immunity. The probiotics and the Chinese herbal medicines supplement each other, the Chinese herbal medicines can promote the proliferation of the probiotics, the probiotics can enhance the absorption efficiency of the Chinese herbal medicines, and the Chinese herbal medicines which are fermented by the probiotics are natural, non-toxic, non-residual, safe and environment-friendly and have wide development prospect.
Compared with the prior art, the Chinese herbal medicine is natural, reliable, safe and environment-friendly, the effective active ingredients in the Chinese herbal medicine are released at an accelerated speed by fermenting the compound Chinese herbal medicine, the taste of the Chinese herbal medicine is improved, the toxicity of the Chinese herbal medicine is reduced, the Chinese herbal medicine is easier to absorb and utilize, the growth promoting effect of probiotics is exerted, the ecological balance of gastrointestinal tracts is adjusted, the appetite of animals is stimulated, the growth of livestock and poultry is promoted, the feed conversion rate is improved, and the immunity of animal organisms is enhanced.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes the immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive for livestock and poultry and the preparation method thereof.
The immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock and poultry mainly comprises a Chinese herbal medicine mixture and probiotics.
The Chinese herbal medicine mixture can comprise, for example: 9-15 parts of folium artemisiae argyi, 6-12 parts of pongamia pinnata, 9-18 parts of hovenia dulcis, 5-15 parts of seaweed and 5-18 parts of elephant grass;
separately, each of the above components may include the following actions: the folium Artemisiae Argyi has antibacterial, antiviral, fetus protecting, reproduction rate increasing, meat quality improving, and weight increasing effects. The Cordia Ellis Bunge has antibacterial, antiinflammatory, analgesic, antiviral, antiulcer and antitumor effects. The hovenia dulcis thunb has the effects of promoting the production of body fluid to quench thirst, clearing heat and relieving restlessness, supplementing nutrition, enhancing disease resistance, tonifying qi and enriching blood. The Sargassum has antiviral, antitumor, mutation resisting, radioprotective, and immunity enhancing effects. The grassiness is excellent forage grass for animals, can improve litter size, enhance body resistance and obviously improve weight gain of fattening pigs.
The raw material components have independent functions, and the raw materials have synergistic interaction, so that the compatibility effect is better than that of a single raw material, for example, the antibacterial activity of the compatibility of the phellodendron amurense rupr and the folium artemisiae argyi is enhanced, and the disease resistance is enhanced; the hovenia dulcis thunb and the elephant grass are matched, so that the crude fiber, the crude protein, the crude fat, the total sugar, the vitamin and other nutrient substances are rich, the proportion is proper, and the weight gain is obvious.
The probiotics provided by the embodiment of the invention can comprise bacillus subtilis and bacillus licheniformis, wherein the bacillus subtilis and the bacillus licheniformis are common probiotics and can be purchased in the market or from the China center for general microorganism culture preservation and management. In addition, the probiotics can also be other probiotics such as lactobacillus plantarum, saccharomyces cerevisiae and the like.
The mixed bacterial suspension of the bacillus subtilis and the bacillus licheniformis is used as one of the raw materials of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock, so that the traditional Chinese medicine fermentation effect can be achieved, the flora composition in livestock can be obviously improved, and the growth of the livestock can be promoted. It is worth to be noted that the two probiotics adopted in the embodiment of the invention have better traditional Chinese medicine fermentation effect than other probiotics, and the double-bacteria fermentation effect is better than the single-bacteria fermentation effect. In addition, the compound has higher stability in the processing process of granules and powder and in an acid environment, and can play a role in the environment in intestinal tracts to proliferate.
In particular, bacillus subtilis and bacillus licheniformis have high stability in the acidic environment of intestinal tracts, and can secrete protease and amylase with strong activities to promote digestion of feed nutrients. Moreover, on the basis of the embodiment of the invention, the two enzymes secreted by the probiotics can dissociate a large amount of effective components existing in the cell walls of the Chinese herbal medicines, thereby being beneficial to the absorption of the organisms of livestock and poultry, enhancing the drug effect, reducing the dosage and solving the problem of slow effect of the Chinese herbal medicines.
In addition, the probiotics used in the embodiment of the invention supplement the Chinese herbal medicine components, the Chinese herbal medicine can promote the proliferation of the probiotics, and the probiotics can enhance the absorption efficiency of the Chinese herbal medicine.
In addition, the embodiment of the invention also provides a preparation method of the fermented Chinese herbal medicine feed additive for enhancing immunity and promoting growth for livestock and poultry, and the preparation method can comprise the following steps: preparing the Chinese herbal medicine mixture into a Chinese herbal medicine fermentation culture medium, inoculating seed liquid of two probiotics into the Chinese herbal medicine fermentation culture medium, and fermenting.
The preparation of seed solution of two probiotics of bacillus subtilis and bacillus licheniformis can comprise the following steps: respectively inoculating Bacillus subtilis and Bacillus licheniformis in seed culture medium, and culturing at 28-37 deg.C for 22-26 hr, preferably at 35 deg.C for 24 hr.
The seed culture medium comprises nutrient broth solid culture medium and nutrient broth liquid culture medium, and the components comprise peptone, beef extract, sodium chloride and the like.
After the seed culture medium is prepared, sterilizing at 121 ℃ for 30-35min, and cooling for later use.
Inoculating is to select the bacillus subtilis and the bacillus licheniformis which are preserved by glycerol, activate the bacillus subtilis and the bacillus licheniformis in the nutrient broth solid culture medium, and then select a single colony which grows well to culture in the nutrient broth liquid culture medium to obtain a seed solution. Preferably, the cultivation is carried out at a rotation speed of 180-220r/min, at which Bacillus subtilis and Bacillus licheniformis, especially Bacillus licheniformis, grow best, and at which too high or too low a rotation speed slows down the growth.
Preferably, the two probiotic strains are cultured in a seed solution according to the weight ratio of 4:6 mixing to obtain mixed bacterial suspension of the bacillus subtilis and the bacillus licheniformis.
Further, the preparation of the Chinese herbal medicine fermentation medium comprises the following steps:
pulverizing the Chinese herbal medicines, uniformly mixing, adding 100-500 parts by weight of water, boiling for 1-3h, then adding glucose, urea, dipotassium hydrogen phosphate, magnesium sulfate and sodium chloride, and uniformly mixing;
optionally, the weight ratio of the Chinese herbal medicine mixture to the glucose, the urea, the dipotassium phosphate, the magnesium sulfate and the sodium chloride is 88-98:2-4:1-2:0.1-0.3:0.01-0.03: 0.3-0.9.
In the embodiment of the invention, the seed liquid of the probiotics inoculated in the Chinese herbal medicine fermentation culture medium accounts for 3-7% of the mass of the Chinese herbal medicine fermentation culture medium. The selection of the inoculum size in the above range ensures a faster fermentation rate. Within this range, the larger the inoculum size, the faster the fermentation rate. Beyond the above inoculation range, the fermentation rate does not increase significantly and the cost increases significantly.
Alternatively, the fermentation in the embodiment of the invention can be carried out for 56-72h at the temperature of 32-36 ℃. Preferably, fermentation is carried out for 64h at 35 ℃.
Because the Chinese herbal medicines used in the embodiment of the invention, such as wampee, seaweed and the like, have certain toxic and side effects if not treated, and are bitter and astringent in taste, the feed intake of animals can be seriously reduced when the Chinese herbal medicines are added into the feed. Therefore, the embodiment of the invention can greatly reduce the bitter taste and the toxic and side effects of the Chinese herbal medicine substances by the fermentation treatment on the premise of ensuring the drug effect, almost has no toxic and side effects, and has the possibility of generating new drug substances.
Further, after fermentation, drying the fermentation product to a moisture content of not more than 10wt%. Drying, and pulverizing the dried product, and sieving with 80-100 mesh sieve.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
(1) Preparing a nutrient broth liquid culture medium with the pH value of 7.2 from 10g/L of peptone, 5g/L of beef extract and 5g/L of NaCl, preparing a nutrient broth solid culture medium from 10g/L of peptone, 5g/L of beef extract, 5g/L of NaCl and 30g/L of agar powder, sterilizing at 121 ℃ for 30min, and cooling to room temperature of 20 ℃ for later use.
(2) Respectively inoculating bacillus subtilis and bacillus licheniformis which are preserved by glycerol into a nutrient broth solid culture medium, activating for 24 hours, picking out single colonies which grow well, respectively inoculating the single colonies into the nutrient broth liquid culture medium, then culturing for 24 hours at the temperature of 35 ℃, and mixing the obtained bacillus subtilis and bacillus licheniformis according to the weight ratio of 4:6 mixing to obtain seed bacteria suspension of the probiotics for later use.
(3) Crushing and uniformly mixing 10 parts by weight of folium artemisiae argyi, 8 parts by weight of pongamia pinnata, 12 parts by weight of hovenia dulcis, 10 parts by weight of seaweed and 10 parts by weight of elephant grass to obtain a Chinese herbal medicine mixture.
(4) Adding 300 parts by weight of water into the Chinese herbal medicine mixture obtained in the step (3), boiling for 2 hours, respectively adding 2 parts by weight of glucose, 1 part by weight of urea, 0.15 part by weight of dipotassium hydrogen phosphate, 0.02 part by weight of magnesium sulfate and 0.4 part by weight of sodium chloride, uniformly mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature of 23 ℃ to obtain the Chinese herbal medicine fermentation medium.
(5) And (3) inoculating the seed bacteria suspension obtained in the step (2) into the Chinese herbal medicine fermentation culture medium obtained in the step (4), wherein the mass of the seed liquid of the probiotics accounts for 5% of the mass of the Chinese herbal medicine fermentation culture medium. Fermenting at 35 deg.C for 72h, oven drying at 60 deg.C until water content is 10wt%, pulverizing, sieving with 80 mesh sieve, and packaging to obtain the fermented Chinese herbal medicine feed additive with immunity enhancing and growth promoting effects for livestock and fowl.
Example 2
(1) Preparing a nutrient broth liquid culture medium with the pH value of 7.4 from 10g/L of peptone, 5g/L of beef extract and 5g/L of NaCl, preparing a nutrient broth solid culture medium from 10g/L of peptone, 5g/L of beef extract, 5g/L of NaCl and 30g/L of agar powder, sterilizing at 121 ℃ for 30min respectively, and cooling to the room temperature of 18 ℃ for later use.
(2) Respectively inoculating bacillus subtilis and bacillus licheniformis which are preserved by glycerol into a nutrient broth solid culture medium, activating for 24 hours, picking out single colonies which grow well, respectively inoculating the single colonies into the nutrient broth liquid culture medium, then culturing for 24 hours at the temperature of 37 ℃, and mixing the obtained bacillus subtilis and bacillus licheniformis according to the weight ratio of 4:6 mixing to obtain seed bacteria suspension of the probiotics for later use.
(3) Crushing and uniformly mixing 10 parts by weight of folium artemisiae argyi, 8 parts by weight of pongamia pinnata, 12 parts by weight of hovenia dulcis, 10 parts by weight of seaweed and 10 parts by weight of elephant grass to obtain a Chinese herbal medicine mixture.
(4) Adding 200 parts by weight of water into the Chinese herbal medicine mixture obtained in the step (3), boiling for 1 hour, respectively adding 1 part by weight of glucose, 0.5 part by weight of urea, 0.05 part by weight of dipotassium phosphate, 0.01 part by weight of magnesium sulfate and 0.2 part by weight of sodium chloride, uniformly mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature of 20 ℃ to obtain the Chinese herbal medicine fermentation medium.
(5) And (3) inoculating the seed bacteria suspension obtained in the step (2) into the Chinese herbal medicine fermentation culture medium obtained in the step (4), wherein the mass of the seed liquid of the probiotics accounts for 7% of the mass of the Chinese herbal medicine fermentation culture medium. Fermenting at 32 deg.C for 70h, oven drying at 60 deg.C until water content is 8wt%, pulverizing, sieving with 80 mesh sieve, and packaging to obtain the fermented Chinese herbal medicine feed additive with immunity enhancing and growth promoting effects for livestock and fowl.
Example 3
(1) Preparing 10g/L peptone, 5g/L beef extract and 5g/L NaCl into a nutrient broth liquid culture medium with the pH value of 7.3, preparing 10g/L peptone, 5g/L beef extract, 5g/L NaCl and 30g/L agar powder into a nutrient broth solid culture medium, sterilizing at 121 ℃ for 30min, and cooling to room temperature of 25 ℃ for later use.
(2) Respectively inoculating bacillus subtilis and bacillus licheniformis which are preserved by glycerol into a nutrient broth solid culture medium, activating for 24 hours, picking out single colonies which grow well, respectively inoculating the single colonies into the nutrient broth liquid culture medium, then culturing for 24 hours at the temperature of 37 ℃, and mixing the obtained bacillus subtilis and bacillus licheniformis according to the weight ratio of 4:6 mixing to obtain seed bacteria suspension of the probiotics for later use.
(3) Crushing and uniformly mixing 10 parts by weight of folium artemisiae argyi, 8 parts by weight of pongamia pinnata, 12 parts by weight of hovenia dulcis, 10 parts by weight of seaweed and 10 parts by weight of elephant grass to obtain a Chinese herbal medicine mixture.
(4) Adding 400 parts by weight of water into the Chinese herbal medicine mixture obtained in the step (3), boiling for 1 hour, respectively adding 1.5 parts by weight of glucose, 1.0 part by weight of urea, 0.1 part by weight of dipotassium hydrogen phosphate, 0.02 part by weight of magnesium sulfate and 0.3 part by weight of sodium chloride, uniformly mixing, sterilizing at 121 ℃ for 35min, and cooling to room temperature of 22 ℃ to obtain the Chinese herbal medicine fermentation medium.
(5) And (3) inoculating the seed bacteria suspension obtained in the step (2) into the Chinese herbal medicine fermentation culture medium obtained in the step (4), wherein the mass of the seed liquid of the probiotics accounts for 3% of the mass of the Chinese herbal medicine fermentation culture medium. Fermenting at 36 deg.C for 54h, oven drying at 60 deg.C until the water content is 6wt%, pulverizing, sieving with 80 mesh sieve, and packaging to obtain the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock and fowl.
Example 4
This example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 13 parts by weight of folium artemisiae argyi, 11 parts by weight of pongamia pinnata, 13 parts by weight of hovenia dulcis thunb, 11 parts by weight of seaweed and 11 parts by weight of elephant grass.
Example 5
This example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 9 weight parts of folium artemisiae argyi, 12 weight parts of pongamia pinnata, 18 weight parts of hovenia dulcis, 12 weight parts of seaweed and 5 weight parts of elephant grass.
Example 6
This example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 15 parts by weight of folium artemisiae argyi, 6 parts by weight of pongamia pinnata, 9 parts by weight of hovenia dulcis thunb, 11 parts by weight of seaweed and 18 parts by weight of elephant grass.
Example 7
This example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 12 parts by weight of folium artemisiae argyi, 8 parts by weight of pongamia pinnata, 15 parts by weight of hovenia dulcis thunb, 15 parts by weight of seaweed and 12 parts by weight of elephant grass.
Example 8
This example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 11 parts by weight of folium artemisiae argyi, 10 parts by weight of pongamia pinnata, 12 parts by weight of hovenia dulcis thunb, 8 parts by weight of seaweed and 15 parts by weight of elephant grass.
Example 9
The present example differs from example 1 in that: the raw materials of the Chinese herbal medicine mixture comprise 15 parts by weight of folium artemisiae argyi, 9 parts by weight of pongamia pinnata, 10 parts by weight of hovenia dulcis, 5 parts by weight of seaweed and 8 parts by weight of elephant grass.
Example 10
This example differs from example 1 in that: the weight ratio of the bacillus subtilis to the bacillus licheniformis is 2:8.
example 11
This example differs from example 1 in that: the weight ratio of the bacillus subtilis to the bacillus licheniformis is 7:3.
example 12
This example differs from example 1 in that: the weight ratio of the bacillus subtilis to the bacillus licheniformis is 5:5.
1. pharmaceutical testing
Test example 1: in vitro bacteriostatic test
The phellodendron amurense extract and the folium artemisiae argyi extract used in the test are both aqueous extracts, and the methicillin-resistant staphylococcus aureus is identified by adopting a cefoxitin paper sheet and a K-B agar paper sheet diffusion method (MRSA is judged if the inhibition zone of cefoxitin to staphylococcus aureus is less than or equal to 19 mm). The minimum inhibitory concentration of the pongamia pinnata extract and the folium artemisiae argyi extract on the experimental strains is determined according to a liquid dilution method recommended by the American clinical laboratory standards Committee. Diluting the medicine by using a 2-time dilution method, wherein the dilution liquid is a sterile culture medium, the concentration of each traditional Chinese medicine extract is 100, 50, 25, 12.5,6.25 and 3.13 mg/mL respectively, 10 mu L of MRSA strain with 0.5 McLeeb's turbidimetric unit is added into each test tube respectively, a blank control is set in the test, the test is carried out at a constant temperature of 37 ℃ for 24h, then common agar plate culture media are respectively transferred, and the concentration of the liquid medicine added into the agar plate culture medium without bacterial growth after 24h is the MRSA-resistant MIC of the traditional Chinese medicine extract. In the experiment, a standard strain staphylococcus aureus ATCC 25923 is used as a quality control strain.
And performing a synergistic antibacterial experiment by adopting a micro-liquid flat chessboard method. The maximum concentration of each traditional Chinese medicine is 2 times of MIC (minimal inhibitory concentration), the traditional Chinese medicine is diluted to 1/16 of the MIC by adopting a multiple ratio dilution method by using an M-H culture medium, 50 mu L of M-H culture medium of two extracts of wampee and folium artemisiae argyi with different concentrations is added along the transverse axis and the longitudinal axis of a sterile micropore plate respectively, and then 1.0 x 10 5 And adding the CFU/mL bacterial liquid into the hole, and simultaneously setting a negative control without adding bacteria and the bacterial liquid without adding any bacteriostatic agent as positive controls. Incubation was carried out at 37 ℃ and the results were observed. MIC of the buffalo hide and the artemisia argyi leaf extract were recorded separately, and MIC of each of the buffalo hide and the artemisia argyi leaf extract when combined for optimal combination effect was selected to calculate fractional inhibitory concentration index (FIC). The test results are shown in Table 1.
FIC Index of refraction =（MIC Buffalo bark combination /MIC Cortex Phellodendri extract ）＋（MIC Folium Artemisiae Argyi combination /MIC Folium Artemisiae Argyi alone ）
Judging the synergistic action of the two medicines: the FIC is less than or equal to 0.5, the FIC is more than 0.5 and less than or equal to 1.0, the FIC is more than 1.0 and less than or equal to 2.0, the additive effect is irrelevant, and the FIC is more than 2.0, the antagonistic effect is obtained.
The experiment investigates the in vitro antibacterial effect of the phellinus igniarius extract and the folium artemisiae argyi extract on methicillin-resistant staphylococcus aureus (MRSA). As a result, it was found that the phellodendron amurense extract and the mugwort leaf extract both have a bactericidal effect on MRSA, but when used alone, a higher concentration is required to be effective, and when combined, the two extracts have FIC Index of refraction Less than or equal to 0.5, and can play a role in synergistic enhancement.
Test example 2: fermentation test of Chinese herbs
Preparing a Chinese herbal medicine fermentation culture medium according to the method in the example 2, respectively inoculating four bacterial suspensions of bacillus subtilis, bacillus licheniformis, lactobacillus plantarum and saccharomyces cerevisiae, wherein the inoculation amount is 6%, standing and fermenting at 37 ℃ for 48 hours, and then determining the content of total flavonoids in the fermentation product. The results of the single-strain fermentation experiments are shown in Table 2.
As can be seen from Table 2, the total flavone content extracted by four probiotics fermentation is greater than that of the unfermented group, and the total flavone content result in the product by four single bacteria fermentation is as follows: bacillus licheniformis, bacillus subtilis, lactobacillus plantarum and Saccharomyces cerevisiae. Therefore, bacillus subtilis and bacillus licheniformis with the highest total flavone content are selected for double-bacterial fermentation, bacterial suspensions of the two bacteria are inoculated according to the following ratio of 9. The results of the two-strain fermentation test are shown in Table 3.
As can be seen from the results in Table 3, the ratio of Bacillus subtilis to Bacillus licheniformis was 4: and 6, the total flavone content after fermentation is highest, and the total flavone content after double-bacterium fermentation is obviously higher than that of single-bacterium fermentation. Therefore, the immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive has better effect by adopting double-bacterium fermentation.
2. Clinical trial
Test example 1: application of immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive in broiler chickens
The immune-enhancing growth-promoting fermented Chinese herbal medicine feed additive in the experiment is the product prepared in the example 1, the example 4 and the example 6, 3000 healthy AA broiler chickens with similar body weight and 1 day old are selected and are randomly divided into 5 groups, each group is divided into 3 times, and each group is divided into 200 times. Wherein the two groups are control groups, wherein the control group 1 is fed with commercial basic diet, and the control group 2 is fed with 0.05% Bacillus subtilis feed additive; tests 1 to 3 groups were carried out by adding 0.5% of the fermented Chinese herbal medicine feed additives of examples 1, 4 and 6 to commercial basal diet. At 35 days of age, randomly extracting 12 chickens with similar body weights from each group, aseptically picking up complete spleen, thymus and bursa of Fabricius immediately after exsanguination and sacrifice of test chickens, sucking surface blood stains, weighing wet mass by using an electronic balance after fat removal, and calculating immune organ indexes of the spleen, the thymus and the bursa of Fabricius. Immune organ index (g/kg) = immune organ weight (g)/live body weight (kg). Weighing at 42 days old, setting up materials, and calculating average daily feed intake, average daily gain and feed conversion rate; the death of the test chickens was recorded throughout the period. The influence of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive on the growth performance of the broilers is shown in table 4, and the influence of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive on the immune organ indexes of the broilers is shown in table 5.
As shown in tables 4 and 5, the test results show that the daily gain and daily feed intake of the broilers in the test group are both higher than those of the broilers in the control group, while the feed meat ratio is lower; the death rate of the broiler chickens in the test group is lower than that of the broiler chickens in the control group; in the aspect of immune organ indexes, the spleen index, the bursa of Fabricius index and the thymus index of the broiler chickens in the control group are higher than those of the broiler chickens in the control group. The immunity-enhancing growth-promoting type fermented Chinese herbal medicine feed additive can improve the utilization rate of the feed, reduce the feed conversion ratio, promote the growth of the broilers and enhance the disease resistance of the broilers.
Test example 2: application of immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive in pigs
A batch of 20-day-old weaned piglets in a certain pig farm in Henan is selected, 600 healthy piglets with similar weight are randomly divided into 5 groups, each group has 3 repetitions, and each repetition has 40 repetitions. Wherein two groups are control groups, wherein the control group 1 is fed with commercial piglet feed as basic ration, and the control group 2 is fed with piglet compound feed composed of commercial piglet feed and 0.1% Bacillus subtilis feed additive; the experiment 1-3 groups are piglet compound feeds prepared by adding 1% of the feed additives of the immune-enhancing and growth-promoting fermented Chinese herbal medicines of the embodiment 1, the embodiment 4 and the embodiment 6 into commercial piglet feeds respectively. The pigs are fed for 21 days under the same feeding management conditions, the hair color of each group is observed during the test period, the indexes of the weight, the daily feed intake, the weight gain, the diarrhea rate and the like of each group are recorded and counted, and the influence of the growth-promoting fermented Chinese herbal medicine feed additive on the growth performance of the pigs is shown in a table 6.
As can be seen from the data in Table 6, the hair color of the pigs in the test group is better than that of the control group, the average daily gain is also higher than that of the control group, the average feed-meat ratio of the test group is 1.28, the feed-meat ratio is reduced by 10 percent compared with that of the control group, and the diarrhea rate is also lower than that of the control group. The experiments prove that the feed additive can improve the feed reward, enhance the immunity of pigs, reduce the occurrence of diseases of piglets and obviously improve the economic benefit of a pig farm.
Test example 3: application of immunity-enhancing growth-promoting fermented Chinese herbal medicine feed additive in ducks
900 healthy cherry valley meat ducks of 1 day old with similar body weight are selected and randomly divided into 5 groups, each group has 3 repetitions, and each repetition has 60 ducks. Wherein the two groups are control groups, wherein the control group 1 is fed with commercial basic diet, and the control group 2 is fed with 0.1% Bacillus subtilis feed additive; tests 1-3 groups were prepared by adding 1% of the growth-promoting, fermented herbal feed additive of example 1, example 4, and example 6 to commercial basal diet, respectively, and the test period was 35 days, weighing and recording every week. Randomly selecting 8 cherry valley meat ducks in each repetition of 14 days and 28 days after feeding, bloodletting and slaughtering jugular veins, weighing thymus, spleen and bursa of fabricius, and calculating immune organ index. Immune organ index (g/kg) = immune organ weight (g)/live body weight (kg). The influence of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive on the growth performance of the meat ducks is shown in table 7, and the influence of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive on the immune organ index of the meat ducks is shown in table 8.
As can be seen from Table 7, the average daily gain and average total gain of the test groups 1 to 3 were significantly higher than those of the control groups 1 and 2, and had significant weight-increasing effects on days 1 to 14 and days 14 to 28, unlike the control group 2 to which Bacillus subtilis was added, which had a weight-increasing effect only on days 14 to 28. In the whole feeding period, the average daily gain and the average total gain of a test group added with the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive are improved by over 60 percent and are improved by over 40 percent compared with the test group added with the bacillus subtilis alone. The results show that the immunity-increasing and growth-promoting fermented Chinese herbal medicine feed additive can improve the weight gain of the cherry valley duck and promote the growth of the cherry valley duck.
As can be seen from Table 8, the thymus index, bursal index and spleen index of the cherry valley ducks of the test groups 1-3 were significantly higher than those of the control groups 1 and 2. On the 28 th day after feeding, the spleen index, the thymus index and the bursa of Fabricius index of the test group added with the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive are respectively improved by more than 30.6 percent, 51.4 percent and 81.7 percent, which are far higher than those of the control group 2 added with only bacillus subtilis. The results show that the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive can stimulate the development of immune organs of cherry valley meat ducks to a certain extent, improve the index of the immune organs and enhance the immune function of organisms, and the results are consistent with the results of the weight-increasing promotion effect of the fermented Chinese herbal medicine feed additive.
The embodiments described above are some, not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (8)
1. A livestock and poultry immunity-enhancing growth-promoting type fermented Chinese herbal medicine feed additive is characterized by comprising a Chinese herbal medicine mixture and probiotics for fermenting the Chinese herbal medicine mixture, wherein the probiotics are used for fermenting the Chinese herbal medicine mixture to prepare a final product;
the Chinese herbal medicine mixture comprises: 9-15 parts of folium artemisiae argyi, 6-12 parts of pongamia pinnata, 9-18 parts of hovenia dulcis, 5-15 parts of seaweed and 5-18 parts of elephant grass; the probiotics comprise 40 parts by weight of bacillus subtilis and 60 parts by weight of bacillus licheniformis; the preparation method of the immunity-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock and poultry comprises the steps of preparing the Chinese herbal medicine mixture into a Chinese herbal medicine fermentation culture medium, inoculating seed liquid of probiotics into the Chinese herbal medicine fermentation culture medium, and fermenting.
2. The livestock and poultry fermented Chinese herbal medicine feed additive capable of enhancing immunity and promoting growth according to claim 1, wherein the Chinese herbal medicine fermentation culture medium is obtained by the following steps:
pulverizing the Chinese herbal medicines, mixing uniformly, adding 100-500 parts by weight of water, boiling for 1-3h, then adding glucose, urea, dipotassium hydrogen phosphate, magnesium sulfate and sodium chloride, and mixing uniformly.
3. The livestock and poultry fermented Chinese herbal medicine feed additive capable of enhancing immunity and promoting growth according to claim 2, wherein the Chinese herbal medicine mixture, the glucose, the urea, the dipotassium hydrogen phosphate, the magnesium sulfate and the sodium chloride are sequentially mixed according to a weight ratio of 88-98:2-4:1-2:0.1-0.3:0.01-0.03: 0.3-0.9.
4. The fermented Chinese herbal medicine feed additive for livestock and poultry, which is used for enhancing immunity and promoting growth, according to claim 1, is characterized in that the seed solution of the probiotics is obtained by the following steps:
respectively inoculating the preserved bacillus subtilis and bacillus licheniformis into a nutrient broth solid culture medium to activate for 22-26h, selecting single colonies with good growth, respectively inoculating into the nutrient broth solid culture medium, then culturing for 22-26h at 28-37 ℃, and mixing to obtain a bacterial suspension of the bacillus subtilis and the bacillus licheniformis; the components of the nutrient broth medium comprise peptone, beef extract and sodium chloride.
5. The immune-enhancing and growth-promoting fermented Chinese herbal medicine feed additive for livestock and poultry according to claim 4, wherein bacillus subtilis and bacillus licheniformis stored in glycerol are respectively inoculated into a nutrient broth solid culture medium for activation; according to the weight ratio of 4:6 mixing the prepared bacterial suspension of the bacillus subtilis and the bacillus licheniformis; culturing at 28-37 deg.C for 22-26h at rotation speed of 180-220 r/min.
6. The fermented Chinese herbal medicine feed additive for livestock and poultry, which is used for enhancing immunity and promoting growth, according to claim 1, is characterized in that the inoculated seed liquid of the probiotics accounts for 3-7% of the mass of the Chinese herbal medicine fermentation culture medium.
7. The fermented Chinese herbal medicine feed additive for livestock and poultry, which is used for enhancing immunity and promoting growth, according to claim 1, is characterized in that the fermentation is carried out for 56-72h at the temperature of 32-36 ℃; after fermentation, drying the fermentation product until the water content is not more than 10wt%; drying, pulverizing the dried product, and sieving with 80-100 mesh sieve.
8. The fermented Chinese herbal medicine feed additive for livestock and poultry, which is used for enhancing immunity and promoting growth, according to claim 1, is characterized in that the fermentation is carried out for 64 hours at 35 ℃.
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