CN100457892C - Traditional Chinese medicine solid type mixed culture fermentation tech. and its application - Google Patents

Traditional Chinese medicine solid type mixed culture fermentation tech. and its application Download PDF

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CN100457892C
CN100457892C CNB2006100388506A CN200610038850A CN100457892C CN 100457892 C CN100457892 C CN 100457892C CN B2006100388506 A CNB2006100388506 A CN B2006100388506A CN 200610038850 A CN200610038850 A CN 200610038850A CN 100457892 C CN100457892 C CN 100457892C
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chinese medicine
fermentation
fermented
mixed
bacterium liquid
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CN1831107A (en
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储卫华
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南京仕必得生物技术有限公司
储卫华
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Abstract

The invention discloses a Chinese drugs multi-bacterium species mixture solid fermenting method and its application. It is made up from crushing the Chinese drugs, filtering, adjusting water content and pH value, adding bean pulp, bran, and inorganic salt, sterilization, cooling and adding bacterium species fermenting liquid to take ferment, and drying. The invention could be used to make drugs and feed additive that has antibiotic, antivirus and growth improvement functions. The invention has the features of simple process method, and improving the growth benefit bacterium.

Description

Many solid type mixed culture fermentations of Chinese medicine method and application thereof
Technical field
The invention belongs to the biological fermentation field, relate to the many solid type mixed culture fermentation tech of a kind of Chinese medicine, the invention still further relates to this technology prepares medicine or green feed additive in livestock and poultry breeding industry application.
Background technology
In today that the economic globalization paces are accelerated, the market access degree constantly enlarges, China's livestock breeding industry has obtained the new opportunity of accelerated development, but also is faced with intense market competition more simultaneously.On the one hand, in the international market, developed country exports animal products to me quality, health, safety etc. require more and more higher, and the threshold of Green Trade Barrier constantly is elevated, and has become the bottleneck of the outlet that is restricting China's animal products; On the other hand, along with the progress of development and national economy and society, the improving constantly of living standards of the people, the domestic consumer not only is satisfied with and has enough to eat and wear, and when paying attention to taste, also will pay attention to the trophic structure and the food safety of various food more.Health, safety, free of contamination food more and more are subjected to each personage's of stratum favor, and the animal products that adopts additives such as microbiotic, growth parahormone to culture will be denied access to market.Under the megatrend of emphasizing food safety day by day, how to improve the animal products quality, to reduce medicine residual, reduce gradually even forbid that antibiotic etc does the use of additive, produce nonreactive food, be the international agriculture market developing trend.In order to seek antibiotic substitute, produce the generation that can effectively prevent livestock and poultry, promote growth of animal, toxic side effect is little, noresidue, the green feed additive that has no drug resistance, the various countries researchist has done a large amount of work, finds that herbal medicine and probiotics (probiotics) are the important developing direction of two classes.
Herbal medicine is the product of distinctive theory of traditional Chinese medical science of China and practice, be that a class has nutrition and medicinal dual function concurrently, possess the function of directly killing or suppressing bacterium and enhancing immunity ability, and can promote the noresidue that nutrient digestion absorbs, the natural drug that has no drug resistance.Aspect the animal doctor, application herbal control livestock and poultry, aquatic animal disease meet the requirement of " safety food production " fully, have both helped human existence health, meet export requirement again, to the great significance of aquaculture.But just the application of herbal medicine in this field at present exists unfavorable factors such as formulation is single, quality is coarse, consumption is big, cost height.For example its formulation just has only Chinese medicine meal and lixiviate.The Chinese medicine meal in use exists can only mixed feeding, and active substance can not be utilized fully like this, has reduced bioavailability, causes dosage to increase.After Chinese medicine undergoes microbial fermentation,,, also produce some biologically active substance simultaneously as lignoenzyme, cellulase, hemicellulase, polygalacturonase etc. because microorganism can produce a large amount of enzymes.Under the effect of these microbial products, Effective Components of Chinese Herb discharges from cell walls, and active constituent content is raise, because the effect of enzyme can produce some new analogues, this finds a good outlet for herbal medicine in the extensive application of aquaculture simultaneously.
The mechanism of action of fermented tcm: enzyme is the biological catalyst that a class has the height catalytic efficiency, and it can make complicated chemical reaction finish rapidly at normal temperatures and pressures.Microorganism can produce various enzymes in process of growth, as cellulase, lignoenzyme, amylase, proteolytic enzyme, lipase etc.These abundant and powerful enzyme systems can form new activeconstituents with the composition decomposition and inversion of medicine, produce new drug effect, fermented tcm can be produced by following approach: at first, microorganism is a precursor with the effective constituent in the Chinese medicine, forms new compound through the metabolism of microorganism; Secondly, microorganism can also produce abundant secondary metabolite in process of growth, and some secondary metabolite self is exactly the effect excellent drug; The 3rd, the secondary metabolite of microorganism and some the material generation chemical reaction in the Chinese medicine generate new compound; The 4th, because some material of Chinese medicine may have promotion or restraining effect to microbial growth and metabolism, generation to activeconstituents also has promotion or restraining effect, microorganism also might change the pathways metabolism of self in the particular surroundings of Chinese medicine, thereby forms new activeconstituents or change the mutual ratio of each activeconstituents; At last, the Decomposition of microorganism might be decomposed the toxic substance in the Chinese medicine, thereby reduces the toxic side effect of medicine, can slough toxic substance as rapeseed cake, cottonseed cake behind microbial fermentation.Utilize the microbial growth metabolism to concoct Chinese medicine, can have more superiority, can change the property of medicine greatly, improve curative effect, reduce toxic side effect, enlarge indication than the process of preparing Chinese medicine means of general physical or chemistry.
Because the effect of microorganism and Chinese medicinal materials is to influence each other in the Chinese medicine fermenting process, and the microorganism majority that adopts is a medicinal fungi at present, the method of fermentation adopts solid-fermented technique more, therefore, part scholar proposes medicinal fungi amphicheirality solid fermentation engineering theory, and the theoretical basis of this technology, the design of fermentation combination, the contents such as pharmacological screening of mycoplasma studied, particular content can be referring to document (Zhuan Yi, medicinal fungi novel (two-way type) solid fermentation engineering. edible fungi of china .2002,21 (4): 3~6).
The key of medicinal fungi novel (amphicheirality) solid fermentation engineering (being called for short two-way fermentation) is that fermented bacterium and fermented substrate are constituted the interior matrix part of fermentation combination, by in the past only with being rich in carbon, the agricultural byproducts of nutrition such as nitrogen (bagasse, wheat bran etc.) as unidirectional nutraceutical matrix (product after its fermentation claims medicinal mycoplasma), change into to adopt and have the Chinese medicinal materials of certain activeconstituents as property of medicine matrix, it can provide the effect of the enzyme that the fungal growth desired nutritional can be subjected to fungi again and be changed structural constituent, thereby produce new nature,taste and action, so have the amphicheirality, product after its fermentation claims medicinal fungal substance, as dual-purpose nutrition, two kinds of matrix of the property of medicine then are called wholeness matrix, and the product after the fermentation also claims medicinal fungal substance.Medicinal fungal substance, might be added with better drug effect simply mutually than this fungi or property of medicine matrix itself or both, mainly show aspects such as synergy, broadened application, detoxifcation (referring to the development of medicinal fungi novel solid fermentation engineering and Chinese scholartree stilbene mycoplasma. Chinese Pharmaceutical Journal, 2004,39 (03), P175~178).
Medicinal fungi novel (amphicheirality) solid fermentation engineering is that traditional Chinese medicine research has been opened up a new field, promptly the fungus medicine (referring to Zhuan Yi. a frontier of Chinese medicine. new Chinese medicine and clinical pharmacology .1992,3 (2) 49~51).But, because the growth conditions of different strain there are differences, each other influence is not fully aware of during many bacterial classifications mixed growth, present research focuses mostly in the research field of single culture to specific Chinese medicinal materials, screening different strain compatibility and technology single or that several kinds of Chinese medicinal materials is fermented are not appeared in the newspapers, and the many solid type mixed culture fermentation tech of herbal medicine are not used for the report of livestock and poultry breeding industry yet.
Summary of the invention:
The objective of the invention is provides a kind of herbal medicine many solid type mixed culture fermentation tech at above-mentioned technical problem.
Another object of the present invention provides above-mentioned solid-fermented technique prepares medicine or green feed additive in livestock and poultry breeding industry application.
The objective of the invention is to realize by following technical measures:
The many solid type mixed culture fermentation tech of a kind of Chinese medicine comprise the following steps:
Preparation fermented by mixed bacterium liquid: two or more original strain is inoculated into production primary seed solution under suitable condition in separately the appropriate media respectively, be transferred to secondary seed solution fermentor tank enlarged culturing at logarithmic phase, fermentation during the secondary seed fermented liquid is transferred to when being cultured to growth and being in logarithmic phase again and produces jar, stop fermentation when reaching logarithmic phase, the bacterium liquid of fermentation respectively is mixed into fermented by mixed bacterium liquid, standby;
The Chinese medicine fermentation: Chinese medicine is pulverized, sieved, be adjusted to suitable water content and pH, add dregs of beans, wheat bran and inorganic salt, sterilization, cooling, inoculation fermented by mixed bacterium liquid, cultivation and fermentation, dry after the fermentation ends.
The many solid type mixed culture fermentation tech of described Chinese medicine, wherein original strain is bacillus cereus (Bacilluscereus), subtilis (Bacillus subtilis), Bacillus licheniformis (Bacilluslicheniformis), lactobacillus lactis (Lactobacillus lactis), Lactobacterium acidophilum (Lactobacillusacidophilus), streptococcus acidi lactici (Streptococcus lactis), high temperature yeast (Candida tropicalis) or geotrichum candidum (Geotrichum candidum).
The many solid type mixed culture fermentation tech of described Chinese medicine, the inoculum size of strain fermentating liquid is the 5-10% of fermented product total mass when wherein carrying out enlarged culturing at different levels.
The many solid type mixed culture fermentation tech of described Chinese medicine, the bacterium liquid equal proportion that adopts each bacterial classification to ferment respectively when wherein preparing fermented by mixed bacterium liquid is mixed into fermented by mixed bacterium liquid.
The many solid type mixed culture fermentation tech of described Chinese medicine, wherein the Chinese medicine fermenting process is that Chinese medicine is pulverized, cross the 80-100 mesh sieve, pack in the fermentor tank, add and account for the dregs of beans of traditional Chinese medicine quality 5%-10%, the wheat bran of 10%-20%, an amount of inorganic salt add water to water content between 60%-70%, regulate pH value 6.0-7.5, feed steam sterilizing 20~25min, cooling adds the mixed strains liquid that accounts for fermented product total mass 5%-10%, and 25~30 ℃ keep 48-96h, at least stir every day 2 times, each 4~6min, fermentation ends, oven dry.
The application of the many solid type mixed culture fermentation tech of described Chinese medicine in livestock and poultry breeding industry.
Described application is medicine or the fodder additives that the many solid type mixed culture fermentation tech preparations of this Chinese medicine of utilization have antibiotic, antiviral and/or growth promoting function in livestock and poultry breeding industry.
The Chinese medicine fermentation preparation or the fodder additives that adopt the present invention to obtain have been compared following advantage with traditional herbal medicine:
1, the protection active ingredient of Chinese herbs exempts from destruction, improves the drug effect of herbal medicine
Microbial fermentation is the bio-transformation of carrying out under comparatively gentle condition such as normal temperature, normal pressure; activeconstituents exempts from destruction in the Chinese medicine so can protect to greatest extent, particularly more can be protected effectively heat sensitive aromatics volatile oil, VITAMIN isoreactivity composition.The expert thinks that fermented tcm is an invention on the Chinese medicine complete processing, be expected to solve Chinese medicine in traditional technologys such as frying in shallow oil, boil, endure, refine, steam, soak, the difficult problem that activeconstituents is difficult to extract to greatest extent.
Comparatively speaking, the molecular weight of fermented tcm is less, thus have in body absorb comparatively fast, fully, characteristic such as result of treatment is better.Medicine enters the effective active component that can not directly be utilized behind the body, it is degraded into small-molecule active substance and is directly utilized by microorganism, can also remove macromole impurity, improves the drug effect of Chinese medicine greatly.Small-molecule active substance thereby has higher activity than macromolecular substance because it is easier to by the barrier of health and the body cell protein binding.
2, produce new drug effect
The preferred animal probiotics itself has the function of replenishing or strengthening original medicine, simultaneously, the enzyme of microorganisms can carry out structural modification to active ingredient of Chinese herbs again, has produced new active substance thereby produce to compare with original medicine, thereby has had new health care, prevention and treatment function.
3, multi-strain fermentation increases the kind of enzyme, promotes the generation of benefit materials
The present invention produces with the many Chinese medicines of multiple zymogenic bacteria kind ferments, because bacterial strain uses therefor can produce as cellulase, proteolytic enzyme, amylase etc., make Mierocrystalline cellulose in the herbal medicine Chinese medicine, xylogen etc. fully to degrade, pharmaceutically active ingredient can become absorbable to greatest extent in not only making, and cellulose conversion can be become oligose, promote the growth of animal gastrointestinal tract probiotics, also biologically active substances such as small molecular protein, total free aminoacids and polypeptide class product are improved greatly simultaneously.
4, Chinese medicine and probiotics use simultaneously, and effect strengthens
The tradition herbal medicine only is that Chinese medicinal materials is directly added in the feed through preliminary pulverizing, effective constituent can not be absorbed by animal or specific absorption extremely low; Or by the extraction of methods such as decoction, its production cost increases.Pass through microbial fermentation technology, make the effective constituent in the Chinese medicinal materials become absorbable main component through bio-transformation, improve important curative effect, the present invention simultaneously selects for use bacterium producing multi enzyme preparation and milk-acid bacteria etc. as fermented bacterium, add in the feed and can replenish enzyme, promote feed to absorb, feed conversion rate is provided, milk-acid bacteria, yeast etc. have the effect of probiotics again in addition.
Embodiment
The invention will be further elaborated by the following examples, the Chinese medicine preparation of the Cai Yonging usefulness for example in order to make those of ordinary skills understand the present invention only wherein unintelligiblely only can be used for this several concrete antibiotic, antiviral, promotes growth sides for method of the present invention.
Bacterial classification used among the embodiment is preserved center and Chinese industrial microorganism preservation center available from Chinese common micro-organisms respectively.Other carbon nitrogen sources that are used for microorganism culturing outside the also available dregs of beans of the carbon nitrogen source that adopts among the embodiment, the wheat bran substitute, the selected growth but this carbon nitrogen source should suit.
Embodiment 1
The antiviral side of Chinese medicine:
Qingwen baidu powder: gypsum 120g, glutinous rehmannia 30g, Cornu Bubali 60g, coptis 20g, cape jasmine 30g, Tree Peony Bark 20g, root of large-flowered skullcap 25g, radix paeoniae rubrathe 25g, radix scrophulariae 25g, wind-weed 30g, capsule of weeping forsythia 30g, balloonflower root 25g, Radix Glycyrrhizae 15g, Herba Lophatheri 25g.
Zymotechnique:
Bacillus cereus DM423 and subtilis CICC10071 are inoculated into broth medium respectively at 37 ℃, and the 250rpm shaking table is cultivated, and the production primary seed solution is when bacteria content reaches 1.0 * 10 7~8During CFU/ml primary seed solution is transferred to secondary seed solution fermentor tank enlarged culturing (ordinary broth), inoculum size is 10% of a fermented product total mass, and culture temperature 25-30 ℃, mixing speed 250rpm works as bacteria content equally and reaches 1.0 * 10 7~8During CFU/ml, inoculate produce jar in 25-30 ℃ of fermentation (as reference, inoculum size is 10% with the fermented product total mass, the dregs of beans of the carbon nitrogen source employing 8% of substratum, 15% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), all the other are water), the preparation strain fermentating liquid is when total plate count reaches 1.0 * 10 9Stop fermentation during CFU/ml, go out jar, the strain fermentating liquid of cultivating is respectively mixed being made into fermented by mixed bacterium liquid by equal proportion, standby;
Prescription by above-mentioned qingwen baidu powder is pulverized Chinese medicinal materials, crosses 100 mesh sieves, and in the fermentor tank of packing into, adding accounts for the dregs of beans of traditional Chinese medicine quality 10%, 20% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), add water to water content between 60%-70%, regulate pH value 7.0 ± 0.3, feed steam sterilizing 20min, cooling adds the fermented by mixed bacterium liquid that accounts for fermented product total mass 10%, and 25-30 ℃ keeps 48h, stirs at least every day 2 times, each 5min.When the bacterium total content reaches 1.0 * 10 6~8Fermentation ends during CFU/g, 60 ℃ of electric heating drying make the Chinese medicine fermentation preparation of qingwen baidu powder.
Embodiment 2
Chinese medicine antibacterium side:
Four flavor Herba Andrographis loose: Herba Andrographis 450g, polygonum flaccidum 150g, Leaf of Indigowoad 200g, Triquetrous Tadehagi Herb 200g.
Zymotechnique:
CICC10098 is inoculated into ordinary broth with Bacillus licheniformis, and lactobacillus lactis CGMCC1.1878 and Lactobacterium acidophilum CGMCC1.9 are inoculated into respectively in the MRS substratum, and 37 ℃, the 250rpm shaking table is cultivated the production primary seed solution, when bacteria content reaches 1.0 * 10 7~8Be transferred to secondary seed solution fermentor tank enlarged culturing during CFU/ml, inoculum size is 10% of a fermented product total mass, 28 ± 2 ℃ of culture temperature, and mixing speed 250rpm, the secondary seed solution bacterial content reaches 1.0 * 10 7~8Be inoculated into during CFU/ml produce jar in 25-30 ℃ of fermentation (as reference, inoculum size is 10% with the fermented product total mass, the dregs of beans of the carbon nitrogen source employing 8% of substratum, 15% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), all the other are water), when total plate count reaches 1.0 * 10 9Stop fermentation during CFU/ml, go out jar, the strain fermentating liquid cultivated respectively is made into fermented by mixed bacterium liquid by 0.8: 1: 1 mixed, standby;
By the prescription that above-mentioned four flavor Herba Andrographis loose Chinese medicinal materials is pulverized, crossed 80 mesh sieves, in the fermentor tank of packing into, add 10% the dregs of beans that accounts for traditional Chinese medicine quality, 15% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), add water to water content between 60%-70%, regulate pH value 6.8 ± 0.3, feed steam sterilizing 20min, cooling adds the fermented by mixed bacterium liquid that accounts for fermented product total mass 10%, and 25-30 ℃ keeps 72h, stirs at least every day 2 times, each 5min.When total plate count reaches 1.0 * 10 6~8Stop fermentation during CFU/g, 65 ℃ of electric heating drying make the Chinese medicine fermentation preparation that four flavor Herba Andrographis loose.
Embodiment 3
Chinese medicine promotes growth side:
Big porker looses: Thickrhizome Wood Fern 30g, Tuber Fleeceflower Root 30g, Fructus Hordei Germinatus 500g, soya bean (stir-fry) 500g.
Zymotechnique:
Subtilis CICC10071, Bacillus licheniformis CICC10098 are inoculated into ordinary broth respectively, lactobacillus lactis CGMCC1.1878 and Lactobacterium acidophilum CGMCC1.9 are inoculated into respectively in the MRS substratum, 37 ℃, the 250rpm shaking table is cultivated the production primary seed solution, when bacteria content reaches 1.0 * 10 7~8CFU/ml is transferred to secondary seed solution fermentor tank enlarged culturing again, and inoculum size is 8% of a fermented product total mass, 28 ± 2 ℃ of culture temperature, and mixing speed 200rpm is when the secondary seed solution bacteria content reaches 1.0 * 10 7~8CFU/ml change be inoculated into produce jar in 25-30 ℃ of fermentation (as reference, inoculum size is 10% with the fermented product total mass, the dregs of beans of the carbon nitrogen source employing 8% of substratum, 15% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), all the other are water), when total plate count reaches 1.0 * 10 9Stop fermentation during CFU/ml, go out jar, with the strain fermentating liquid cultivated respectively by 1: 0.8: 1.1: 1 mixed is made into fermented by mixed bacterium liquid, and is standby;
By the prescription that above-mentioned big porker looses Chinese medicinal materials is pulverized, crossed 90 mesh sieves, in the fermentor tank of packing into, add 5% the dregs of beans that accounts for traditional Chinese medicine quality, 20% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), add water to water content between 60%-70%, regulate pH value 6.8 ± 0.3, feed steam sterilizing 20min, cooling adds the fermented by mixed bacterium liquid that accounts for fermented product total mass 8%, and 25-30 ℃ keeps 96h, stirs at least every day 2 times, each 5min.When total plate count reaches 1.0 * 10 6~8Stop fermentation during CFU/g, 55 ℃ of electric heating drying make the Chinese medicine fermentation preparation that big porker looses.
Embodiment 4
Chinese medicine antiviral growth-promoting side:
Strong chicken looses: Radix Codonopsis 20g, Radix Astragali 20g, Poria cocos 20g, Medicated Leaven 10g, Fructus Hordei Germinatus 10g, hawthorn 10g, Radix Glycyrrhizae 5g, betel nut 5g
Zymotechnique:
Streptococcus acidi lactici CGMCC1.1936, high temperature yeast CGMCC2.617 and geotrichum candidum CGMCC2.498 are inoculated into respectively in MRS, YPD, the malt extract medium, streptococcus acidi lactici is at 37 ℃, 30 ℃ of high temperature yeast and geotrichum candidums, the 250rpm shaking table is cultivated the production primary seed solution, when content of microorganisms reaches 1.0 * 10 6~8Be transferred to secondary seed solution fermentor tank enlarged culturing during CFU/ml, inoculum size is 5%, 28 ± 1 ℃ of culture temperature, and mixing speed 230rpm, the secondary seed solution content of microorganisms is 1.0 * 10 6~8Be inoculated into during CFU/ml produce jar in 28 ± 1 ℃ of fermentations (as reference, inoculum size is 10% with the fermented product total mass, the dregs of beans of the carbon nitrogen source employing 8% of substratum, 15% wheat bran, an amount of inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), all the other are water, when microbial count reaches 1.0 * 10 9Stop fermentation during CFU/ml, go out jar, the strain fermentating liquid of cultivating is respectively mixed being made into fermented by mixed bacterium liquid by equal proportion, standby;
By the prescription that above-mentioned strong chicken looses Chinese medicinal materials is pulverized, crossed 80 mesh sieves, in the fermentor tank of packing into, add 10% the dregs of beans that accounts for traditional Chinese medicine quality, 10% wheat bran, 0.5% sucrose, inorganic salt (MnSO 40.1%, K 2HPO 40.3%, KH 2PO 40.15%, MgSO 40.05%, CaCO 30.01%), add water to water content between 60%-70%, regulate pH value 7.2 ± 0.2, feed steam sterilizing 20min, cool off, account for the fermented by mixed bacterium liquid of fermented product total mass 5%, 25-30 ℃ keeps 96h, stirs at least every day 2 times, each 5min.When microbial count reaches 1.0 * 10 6~8Stop fermentation during CFU/g, 70 ℃ of electric heating drying make the Chinese medicine fermentation preparation that strong chicken looses.
Embodiment 5
Many bacterial classifications of Chinese medicine solid fermentation preparation is to fryer partial immunity index and somatotrophic influence
1 material
1.1 medicine and reagent
Chinese medicine ordinary preparation: strong chicken diffusing (Radix Codonopsis 20g, Radix Astragali 20g, Poria cocos 20g, Medicated Leaven 10g, Fructus Hordei Germinatus 10g, hawthorn 10g, Radix Glycyrrhizae 5g, betel nut 5g).Take by weighing above-mentioned raw materials and pulverize, cross 80 orders promptly.
The Chinese medicine fermentation preparation: fermenting by embodiment 4 makes.
Newcastle disease IV is vaccine (production of Zhongmu Stocks Trading Co. Nanjing medical instruments factory).
1.2 experimental animal: 90 of 1 age in days Ai Wei mattress chicks for meat, Nanjing kind Green Dragon pheasant factory provides.90 1 age in days Ai Wei mattress fryer are divided into 3 groups, 30 every group at random.Offer medicine twice respectively when 2~6 ages in days and 25~30 ages in days, the consumption that the A group is pressed forage volume 1% adds the Chinese medicine ordinary preparation, and the B group is added the Chinese medicine fermentation preparation by 1% consumption, and the blank group of C group for not adding any medicine raised according to a conventional method.
1.3 feeding effect detects index
1.3.1 newcastle epidemic disease antibody level detection: is that vaccine carries out routine immunization in 7,21 ages in days with newcastle disease IV, detects anti-newcastle disease vaccine antibody horizontal respectively when 20 and 35 ages in days.The blood clotting of the NDV that measuring method employing β-micromethod is measured suppresses (HI) antibody, and the result represents with log2.
1.3.2 immune organ assessment of indices: slaughter all chickens when raising, gather spleen, the fabricius bursa to 45 ages in days, draw the moisture on surface with filter paper after, weigh with precision balance, according to calculating the organ index of spleen, the fabricius bursa with the ratio of physique amount.
1.3.3 chicken physique amount: weigh respectively at 1,7,14,21,28,35,45 ages in days, observe each stage chicken growth of weight situation.
2 results
2.1 hemagglutination inhibition antibody is tired after each test group newcastle disease vaccine immunity, the results are shown in Table 1.As shown in Table 1, compare significant difference (P<0.05) with the blank group at the antibody horizontal of for the second time immune (21 age in days) preceding A, B group chicken.B group antibody horizontal is organized significant difference (P<0.05) apparently higher than A after immunity for the second time.Show that fermentation preparation is being better than not fermentation preparation aspect the humoral immunization that improves body.
Table 1 is respectively organized newcastle disease HI antibody titer (X ± S)
Annotate: the right expression significant difference that indicates different letters of takeing on of colleague's numeral, capitalization represents that difference is extremely remarkable, the lowercase alphabet differential is different significantly, down together.
2.2 the immune organ index is by table 2 as seen, A, B group fabricius bursa exponential sum index and spleen index is organized apparently higher than C, significant difference (P<0.05), and the B group is compared significant difference (P<0.05) with the A group.Test shows that 2 test group preparations all have the effect that promotes growth to spleen and these two immune organs of the fabricius bursa, thereby Abwehrkraft des Koepers is strengthened, and the most outstanding with the fermentation preparation group.
Table 2 fabricius bursa exponential sum index and spleen index (X ± S)
2.3 the influence to meat chicken production performance: as shown in Table 3, initial physique amount does not have significant difference between 2 test group, delivers the physique amount for sale and compares significant difference (P<0.05) with feedstuff-meat ratio A, B group with the C group.The B group is compared significant difference (P<0.05) with A, C group.Show this Chinese medicine preparation by fermentation, obviously improved efficiency of feed utilization, can reduce the usage quantity of simple Chinese medicine simultaneously.
Table 3 body weight gain situation and feedstuff-meat ratio (X ± S)
Conclusion: Chinese medicine preparation by fermentation after, the hemagglutination inhibition antibody of anti-newcastle disease is tired, the immune organ index be higher than the fermentation before preparation, gaining effect (efficiency of feed utilization) is also very obvious.
Embodiment 6
Many bacterial classifications of Chinese medicine solid fermentation preparation is to the external bacteriostatic action of bacterium
1, material
1.1 medicament
Chinese medicine ordinary preparation: four flavor Herba Andrographis diffusing (Herba Andrographis 450g, polygonum flaccidum 150g, Leaf of Indigowoad 200g, Triquetrous Tadehagi Herb 200g).
Chinese medicine fermentation preparation: by embodiment 2 above-mentioned Chinese medicine preparation is fermented and to make.
1.2 bacterial classification and culture condition: intestinal bacteria, streptococcus aureus, subtilis, pasteurella multocida are provided by Agricultural University Of Nanjing's veterinary microorganism group, and experimental bacteria is inoculated in 37 ℃ of cultivation 18~24h in the common nutrient broth, and be standby.
2, method
2.1 the preparation of traditional Chinese medicine liquid: make up a prescription by prescription, respectively take by weighing 100g after the oven dry, add water 1~2L, soak 30min; Decoct, boil back 30min at a simmer, with 4 layers of filtered through gauze soup; The dregs of a decoction add water 0.5~1L, fry in shallow oil 30min after boiling, filtering liquid medicine; Merge 2 times soup, be concentrated into 100mL (crude drug content is 1g/mL in the soup) with slow fire, it is standby to sterilize.The Chinese medicine fermentation preparation is by being prepared into solution with quadrat method.
2.2 test method: adopt 2 times of dilution methods of test tube to measure minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of each bacterium.Every kind of medicine is all used 10 gradients of 2 times of serial dilutions of broth culture in the test, and the 11st test tube made positive control, and the 12nd pipe is made negative control, and the final volume after each pipe dilution is 2mL.Every pipe adds the broth culture of test organisms, and (extent of dilution is 10 -3) 0.05mL, put 37 ℃ and cultivate 16~18h, whether observe has bacteria growing.Every kind of medicine is done 2 groups of repetitions.
2.3 the result judges: get the test organisms nutrient solution that contains each extent of dilution herbal medicine, adopt the cotton swab method of scoring to be inoculated in plain agar plate culture medium surface, put 37 ℃ and cultivate 24h.The minimum drug level of no bacterial growth is counted the MIC (get 2 group repeat mean value) of this medicine to test organisms.The nutrient agar plate of not growing bacterium is continued to cultivate 24h, observe the growing state of bacterium, not grow the minimum concentration of bacterium, as the MBC of medicine.
3 results
Chinese medicine ordinary preparation and fermentation preparation see Table 4 to the vitro inhibition effect of common bacteria.
Table 4 Chinese medicine ordinary preparation and fermentation preparation are to the MIC and the MBC value of common bacteria
Conclusion: after Chinese medicine preparation was handled by fermentation, its anti-microbial activity obviously strengthened.

Claims (4)

1, a kind of many solid type mixed culture fermentations of Chinese medicine method is characterized in that comprising the following steps:
Preparation fermented by mixed bacterium liquid: two or more original strain is inoculated into production primary seed solution under suitable condition in separately the appropriate media respectively, be transferred to secondary seed solution fermentor tank enlarged culturing at logarithmic phase, fermentation during the secondary seed fermented liquid is transferred to when being cultured to growth and being in logarithmic phase again and produces jar, stop fermentation when reaching logarithmic phase, the bacterium liquid of fermentation respectively is mixed into fermented by mixed bacterium liquid, standby;
Chinese medicine fermentation: Chinese medicine is pulverized, crossed the 80-100 mesh sieve, in the fermentor tank of packing into, adding accounts for the dregs of beans of traditional Chinese medicine quality 5%-10%, the wheat bran of 10%-20%, and an amount of inorganic salt add water to water content between 60%-70%, regulate pH value 6.0-7.5, feed steam sterilizing 20~25min, cooling, add the mixed strains liquid that accounts for fermented product total mass 5%-10%, 25~30 ℃ keep 48-96h, stir each 4~6min every day at least 2 times, fermentation ends, oven dry;
Wherein: original strain is bacillus cereus, subtilis, Bacillus licheniformis, lactobacillus lactis, Lactobacterium acidophilum, streptococcus acidi lactici, high temperature yeast or geotrichum candidum.
2, many solid type mixed culture fermentations of Chinese medicine method according to claim 1, the bacterium liquid equal proportion that adopts each bacterial classification to ferment respectively when it is characterized in that preparing fermented by mixed bacterium liquid is mixed into fermented by mixed bacterium liquid.
3, the application of many solid type mixed culture fermentations of the described Chinese medicine of claim 1 method in livestock and poultry breeding industry.
4, application according to claim 3 is characterized in that this many solid type mixed culture fermentations of Chinese medicine method of utilization prepares medicine or the fodder additives with antibiotic, antiviral and/or growth promoting function in livestock and poultry breeding industry.
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