阅读说明：本技术 一种鸡粪菌发酵黄芩茎叶水煎药渣制备的禽用饲料添加剂及其制备方法和应用 (Poultry feed additive prepared from chicken manure bacteria fermented scutellaria baicalensis stem and leaf water decoction dregs and preparation method and application thereof ) 是由 王欢 张希春 胡会玲 武怡荷 于 2020-04-15 设计创作，主要内容包括：本发明属中兽药与中药饲料添加剂领域,为解决目前黄芩茎叶利用度以及利用方式存在的问题,提供一种鸡粪菌发酵黄芩茎叶水煎药渣制备的禽用饲料添加剂及其制备方法和应用。用高纤维压力在鸡胃肠的模拟环境条件下筛选高β-葡萄糖苷酶活性的鸡源益生菌群,用筛选的高β-葡萄糖苷酶活性的鸡源益生菌群发酵黄芩茎叶水煎后的药渣,干燥,与水煎浓缩液混合后干燥、粉碎、制粒得到为禽用饲料添加剂。添加发酵型黄芩茎叶原煎鸡饲料添加剂使40-42日龄、62-64日龄肉鸡对日粮干物质、粗蛋白和粗灰分的消化利用,表观消化率显著高于未发酵添加组。使用一定量的发酵型黄芩茎叶原煎鸡饲料添加剂有提高肉鸡、蛋鸡生产性能,增加抗病能力等有益作用。(The invention belongs to the field of traditional Chinese veterinary medicines and traditional Chinese medicine feed additives, and provides a poultry feed additive prepared by fermenting scutellaria baicalensis stem and leaf water decoction dregs with coprophila, a preparation method and an application thereof, aiming at solving the problems of the availability and utilization mode of scutellaria baicalensis stem and leaf at present. Screening chicken-derived probiotic groups with high beta-glucosidase activity under simulated environment conditions of chicken intestines and stomach by high fiber pressure, fermenting dregs obtained after decocting scutellaria baicalensis stem leaves with water by using the screened chicken-derived probiotic groups with high beta-glucosidase activity, drying, mixing with the decoction concentrate, drying, crushing and granulating to obtain the poultry feed additive. The fermented scutellaria baicalensis stem and leaf primary-decocted chicken feed additive is added, so that the broiler chickens of 40-42 days old and 62-64 days old can digest and utilize the dry matter, crude protein and crude ash of the daily ration, and the apparent digestibility is obviously higher than that of the unfermented additive group. The feed additive for the original fried chicken with the fermented scutellaria baicalensis stem leaves has the beneficial effects of improving the production performance of broiler chickens and laying hens, increasing the disease resistance and the like.)

1. A poultry feed additive prepared from chicken manure bacteria fermented scutellaria baicalensis stem and leaf water decoction dregs is characterized in that: the poultry feed additive is prepared by screening chicken-derived probiotic groups with high beta-glucosidase activity under simulated environment conditions of chicken intestines and stomach by using high fiber pressure, fermenting dregs obtained after water decoction of scutellaria baicalensis stem leaves by using the chicken-derived probiotic groups with high beta-glucosidase activity, drying the fermented dregs, and crushing.

2. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 1 is characterized in that: the method for screening the chicken-derived probiotic flora with high beta-glucosidase activity specifically comprises the following steps:

1) treating a manure sample: adding 5g of chicken manure sample of a free-range healthy laying hen into 45ml of sterile water, uniformly mixing at constant temperature of 37 ℃ under 110 r/min in a shaking way, centrifuging at 3000 r/min for 10-20 min, and taking a bacterial suspension for later use;

2) the chicken simulated gastrointestinal system is combined with the high fiber pressure formed by the bran culture medium to screen the chicken-derived probiotic flora with high beta-glucosidase activity:

chicken simulated gastrointestinal system: the chicken stomach and intestine simulating system consists of a culture tube containing a gastric juice and bran culture medium, simulated small intestine liquid is added into the original culture tube after the stomach simulation is finished, and the pH value is adjusted to form a simulated small intestine environment;

1mL of chicken dung bacteria suspension is put in a chicken simulated gastrointestinal system containing a bran culture medium, and is respectively subjected to shaking culture for 24 hours, 48 hours and 96 hours at the temperature of 37 ℃ and at the speed of 110 r/min; after the culture is finished, centrifuging at 3000 r/min for 10-20 min, taking 1mL of bacterial suspension, placing in an enrichment medium, keeping the temperature at 37 ℃, carrying out shaking culture at 110 r/min for 12-48h, and storing for later use.

3. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 2 is characterized in that: the simulated gastric juice formula comprises: NaCl 2g, pepsin 3.2g, 36.5% HCl 7mL, sterile distilled water to constant volume of 100 mL;

the simulated intestinal juice formula comprises: trypsin 0.9g, bile salt 6g, NaHCO₃12.5g, dissolving in sterile distilled water and fixing the volume to 1000 mL;

the formula of the bran culture medium is as follows: bran 4.0%, urea 0.2%, (NH)₄)₂SO₄0.2%，MgSO₄·7H₂O 0.05%，KH₂PO₄Sterilizing at 115 deg.C for 30min at 0.1%;

the formula of the enrichment medium is as follows: cellobiose 0.5%, streptomycin 0.02%, (NH)₄)₂SO₄0.3%，MgSO₄·7H₂O0.05%，KH₂PO₄0.1 percent, NaCl 0.2 percent and sterilized for 30min at 115 ℃.

4. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 2 is characterized in that: the healthy laying hens comprise: no medicine or antibiotic is used within 3 months, and the energy provided by the concentrated feed accounts for less than or equal to 50 percent of the daily total energy requirement.

5. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 1 is characterized in that: the specific treatment method of the scutellaria baicalensis leaves comprises the following steps: soaking stem and leaf of Scutellariae radix in 360L water for 1 hr, decocting for 50min, separating the liquid by hot press filtration, drying the residue at 85 deg.C for 30-45min, and concentrating the liquid to 5-10L.

6. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 1 is characterized in that: the specific method for decocting the residue of the chicken-derived probiotic flora fermented scutellaria baicalensis stem and leaf with water comprises the following steps: mixing the water-decocted concentrated solution of the stem and leaf of the scutellaria with the dried and filter-pressed dregs of the stem and leaf of the scutellaria, and enabling the dregs to fully and uniformly absorb the liquid medicine to form a solid culture medium matrix of the stem and leaf dregs of the scutellaria;

inoculating 1-3% of the bacterial flora with high beta-glucosidase activity into a rich culture medium, keeping the temperature at 37 ℃, and carrying out shaking culture at 110 r/min for 12-48h to obtain a revived bacterial suspension; mixing 1-5L of revived bacterial suspension with 100Kg of Scutellaria baicalensis stem and leaf residue solid culture medium, and performing solid fermentation at normal temperature for 10-30 days.

7. The poultry feed additive prepared from the coprophilus albuminosus fermented scutellaria baicalensis stem and leaf water decoction dregs according to claim 6, is characterized in that: the formula of the rich culture medium is as follows: cellobiose 0.5%, streptomycin 0.02%, (NH4)2SO40.3%, MgSO4 & 7H2O 0.05%, KH2PO40.1%, NaCl 0.2%, sterilizing at 115 deg.C for 30 min;

the formula of the scutellaria baicalensis stem and leaf decoction dreg solid culture medium is as follows: 99Kg of culture medium matrix of scutellaria stem and leaf dregs solid, 400g of urea, (NH4)2SO 4400 g, 50g of MgSO4 & 7H2O 50 and KH2PO 4150 g.

8. The poultry feed additive prepared from the coprophilus albuminosus fermented scutellaria baicalensis stem and leaf water decoction dregs according to claim 6, is characterized in that: and performing normal-temperature solid fermentation on the stem and leaf of the scutellaria baicalensis at the end of autumn in the harvesting period.

9. The poultry feed additive prepared from the coprophilous-coprinus-fermented scutellaria baicalensis stem-leaf water decoction dregs according to claim 1 is characterized in that: the concrete method for obtaining the poultry feed additive by molding comprises the following steps: drying the solid fermented crude scutellaria stem and leaf decoction dregs at 65 ℃ for 30-90min, then crushing the dregs into coarse powder without sieving, mixing 1kg of the crushed dregs with 1-3L of water decoction concentrated solution, then drying the mixture at 65 ℃ for 30-90min, crushing the mixture, sieving the dried mixture with a 40-mesh sieve, and sealing and packaging the crushed mixture to obtain the fermented crude scutellaria stem and leaf decoction poultry feed additive.

Technical Field

The invention belongs to the field of traditional Chinese veterinary medicines and traditional Chinese medicine feed additives, and particularly relates to a poultry feed additive prepared by fermenting scutellaria baicalensis stem and leaf water decoction dregs with coprophilous dung, and a preparation method and application thereof.

Background

The clinical dosage of the scutellaria baicalensis is large, wild resources are gradually deficient, artificial cultivation resources become main sources of the scutellaria baicalensis, and the cultivation area and the yield are increasingly increased. During the process of collecting scutellaria root, a large amount of non-traditional medicinal parts such as overground part of scutellaria including overground stem, leaf and flower are generated, the annual output is about 3 times of the root output, and most of the overground part is not effectively utilized and discarded, so that a large amount of resource waste and environmental pollution are caused.

The young leaves and stems of scutellaria baicalensis are drunk as tea leaves in northern provinces and Yunnan parts of China, are named as scutellaria baicalensis tea, golden tea and the like, are prepared by collecting, selecting, cleaning, steaming at high temperature, deactivating enzymes, kneading, baking, frying, extracting fragrance and other procedures of the young stems and leaves of overground parts, have the functions of clearing heat and drying dampness, purging fire and detoxifying, diminishing inflammation, promoting digestion and the like^［1］. The stem and leaf of scutellaria baicalensis contains rich resource chemical components, mainly contains flavones, volatile oil, diterpenes, phenolic acids, nucleotide amino acids and inorganic elements, the types of the elements are almost the same as those of the root, but the content difference is larger, and the content of scutellarin and phenolic acids in the stem and leaf is obviously higher than that of the root. The scutellaria stem and leaf coarse powder water extract is subjected to reduced pressure concentration, centrifugal macroporous resin purification, eluent and drying to obtain the scutellaria stem and leaf total flavone, and the scutellaria stem and leaf detoxification capsules taking the scutellaria stem and leaf coarse powder water extract as the main component are approved to be sold on the market and are clinically used as anti-inflammatory drugs^［2-5］。

Search of patent application related to the invention patent of the overground part of scutellaria baicalensis: 1 method for extracting and separating high-purity wild rhubarb and other glycosides from scutellaria stem and leaf; a scutellaria stem and leaf powder injection and a preparation method thereof; the application of stem and leaf of Scutellaria baicalensis Georgi in preparing medicine for preventing and treating virus and/or bacterial infection; extracting total flavone from stem and leaf of Scutellariae radix; a method for preparing scutellaria drinking water by using the effective components of the stem, leaf and flower of scutellaria; tea bag of stem and leaf of radix Scutellariae; the application of total flavonoids of stem and leaf of Scutellariae radix and scutellarin thereof in preparing anti-dementia medicine is provided; the application of the stem and leaf of scutellaria in preparing analgesic, anti-inflammatory and/or antipyretic medicine; application of stem and leaf of Scutellariae radix in preparing medicine is provided.

Therefore, the patent quantity of the stem and leaf of the scutellaria baicalensis is less, the application range of the stem and leaf of the scutellaria baicalensis is basically concentrated on medicines and foods, and the application of the flavonoid compound in the stem and leaf of the scutellaria baicalensis and the monomer scutellarin thereof in the aspects of preventing and treating viruses, resisting bacteria, relieving fever and easing pain is mainly utilized

The application status of the fermented traditional Chinese medicine in poultry industry: the broad traditional Chinese medicine fermentation refers to the biotransformation of traditional Chinese medicines and effective components thereof by microorganisms, enzymes and biological cells, and the narrow traditional Chinese medicine fermentation refers to the fermentation of traditional Chinese medicines and effective components thereof by applying microorganisms, aiming at obtaining new effective components of traditional Chinese medicines, improving the release of the current effective components, improving the safety of the traditional Chinese medicines, and exploring the action mechanism and metabolic process of the traditional Chinese medicines^［6］。

The fermented traditional Chinese medicine has important functions in improving the production performance and immunity of poultry and preventing and treating diseases. The fermented traditional Chinese medicine can improve feed intake, promote weight gain, reduce feed conversion ratio, improve laying rate, improve meat quality or egg quality and the like; can improve immune organ index, antibody level, lymphocyte number, etc. The fermentation process of traditional Chinese medicine can be divided into liquid fermentation and solid fermentation, wherein the liquid fermentation usually utilizes single or mixed strain liquid fermentation of aqueous extract of single or compound Chinese herbal medicine^［7-10］. The solid fermentation is carried out by preparing powder from single or compound Chinese herbal medicines, and fermenting with single or mixed strain, such as Massa Medicata Fermentata^［11］Bacillus, Candida gastral and Lactobacillus acidophilus^［12］White rot fungi and yeasts^［13］The solid fermentation is also carried out by using dregs^［14］。

Although fermented traditional Chinese medicines including scutellaria baicalensis are frequently applied in animal husbandry and resource utilization of scutellaria baicalensis stems and leaves is frequently reported, research reports on fermentation utilization of scutellaria baicalensis stems and leaves or decoction dregs obtained after water decoction of scutellaria baicalensis stems and leaves are not reported yet.

The resource utilization of the stem and leaf of scutellaria baicalensis has been a focus of attention in animal husbandry, the purpose of utilizing the stem and leaf of scutellaria baicalensis can be achieved by the traditional decocting and extracting process of traditional Chinese medicines, but certain problems still exist in the aspects of full utilization degree and proper utilization mode, and the two aspects are mainly shown. Firstly, dregs of decoction of the stem and leaf of the scutellaria baicalensis are not fully utilized, which is a certain resource waste; secondly, the utilization mode of the scutellaria stem and leaf decoction is still limited to the drinking water utilization of concentrated solution or the feed mixed feeding mode of formula granules, and is complex and has serious loss of effective components.

Reference documents:

[1] the application history of scutellaria tea and the current situation of research make internal disorder or usurp [ several modern chinese traditional medicines, 2011, 13 (6): 3-7.

[2] UPLC fingerprint and chemical pattern recognition research make internal disorder or usurp [ J ] of stem and leaf of Scutellaria baicalensis in different producing areas, namely Baical skullcap root, Baical: 633-638.

[3] Zhang yu, wu bin research development of pharmacological effects of scutellaria [ J ] medical review, 2013, 19 (6): 1091-1093.

[4] Chinese medicinal materials company, Chinese resources of Chinese traditional medicine [ M ] science publishers, 1995.

[5] The application history of scutellaria tea and the current situation of research make internal disorder or usurp [ several modern chinese traditional medicines, 2011, 13 (6): 3-7.

[6] Wuli Ke, progress of fermented traditional Chinese medicine and traditional Chinese medicine fermentation research make internal disorder or usurp [ J ] the third special medicine and Shandong-Henan-Hubei Sanzhou combined microecology academic conference argument, 2011: 4-11.

[7] Lactobacillus fermentation strengthening and detoxifying powder composite preparation and its application in broiler chicken are make internal disorder or usurp [ J ]. Chinese animal husbandry, 2016, (04): 1066-1071.

[8] Tangyanhong, a novel biological traditional Chinese medicine basic research make internal disorder or usurp, a preparation technology and application thereof on broilers [ D ]. Chinese agriculture university, 2005.

[9] The Chinese herbal medicine preparation affects the production performance and serum biochemical indexes of laying hens before and after fermentation [ J ]. Shandong livestock veterinarians, 2016 (09): 2-4.

[10] Korean populus, liu cui yan, cow bells, changes in ingredients before and after fermentation of Chinese herbal preparations and effects on partial immune indicators and growth of broilers [ J ]. reports on veterinary medicine for livestock husbandry, 2005, (11): 116-120.

[11] Influence of fermented traditional Chinese medicine additives on immune function and antioxidant function of broiler [ J ] veterinary report of livestock husbandry, 2015, (05): 863-867.

[12] Zhang Gui Zhi, Chen Ying, junk and double star, influence of the mixed fermentation preparation of compound traditional Chinese medicine on immune function of AA broiler [ J ]. Chinese veterinary magazine, 2016 (07): 52-55.[7]

[13] Yan Min Dynasty influence of bio-fermented traditional Chinese medicines on the later-stage production performance of egg laying hens [ J ]. Chinese poultry, 2015, (16): 62-63.

[14] Yan surpassing the influence of the astragalus root dregs fermentation preparation on the growth performance of green foot partridge chicken and the biochemical indexes of partial serum [ D ]. Anhui agricultural university, 2016.

Disclosure of Invention

The invention provides a poultry feed additive prepared by fermenting scutellaria baicalensis stem and leaf water decoction dregs with coprophilous dung to solve the problems of availability and utilization mode of scutellaria baicalensis stem and leaf at present, and a preparation method and application thereof.

The invention is realized by the following technical scheme: a poultry feed additive prepared from chicken dung bacteria fermented scutellaria baicalensis stem and leaf water decoction dregs is characterized in that chicken source probiotics groups with high beta-glucosidase activity are screened under the simulated environment condition of chicken intestines and stomach by utilizing high fiber pressure, the chicken source probiotics groups with high beta-glucosidase activity obtained by screening are utilized to ferment drug dregs obtained after scutellaria baicalensis stem and leaf water decoction, then the fermented medicine dregs are dried, mixed with water decoction concentrated solution, dried, crushed and granulated to obtain the poultry feed additive.

The method for screening the chicken-derived probiotic flora with high beta-glucosidase activity specifically comprises the following steps:

1) treating a manure sample: adding 5g of chicken manure sample of a free-range healthy laying hen into 45ml of sterile water, uniformly mixing at constant temperature of 37 ℃ under 110 r/min in a shaking way, and centrifuging at 3000 r/min for 10-20 min to obtain a bacterial suspension for later use;

2) chicken stomach and intestine simulating system and high fiber pressure are combined to screen chicken source probiotics with high beta-glucosidase activity:

chicken simulated gastrointestinal system: the chicken stomach and intestine simulating system consists of a culture tube containing a gastric juice and bran culture medium, simulated small intestine liquid is added into the original culture tube after the stomach simulation is finished, and the pH value is adjusted to form a simulated small intestine environment;

1mL of chicken dung bacteria suspension is put in a chicken simulated gastrointestinal system containing a bran culture medium, and is respectively subjected to shaking culture for 24 hours, 48 hours and 96 hours at the temperature of 37 ℃ and at the speed of 110 r/min; after the culture is finished, centrifuging at 3000 r/min for 10-20 min, taking 1mL of bacterial suspension, placing in an enrichment medium, keeping the temperature at 37 ℃, carrying out shaking culture at 110 r/min for 12-48h, and storing for later use.

Wherein: the simulated gastric juice formula comprises: NaCl 2g, pepsin 3.2g, 36.5% HCl 7mL, sterile distilled water to constant volume of 100 mL;

the simulated intestinal juice formula comprises: trypsin 0.9g, bile salt 6g, NaHCO₃12.5g, dissolving in sterile distilled water and fixing the volume to 1000 mL;

the formula of the bran culture medium is as follows: bran 4.0%, urea 0.2%, (NH)₄)₂SO₄0.2%，MgSO₄·7H₂O 0.05%，KH₂PO₄Sterilizing at 115 deg.C for 30min at 0.1%;

the formula of the enrichment medium is as follows: cellobiose 0.5%, streptomycin 0.02%, (NH)₄)₂SO₄0.3%，MgSO₄·7H₂O0.05%，KH₂PO₄0.1 percent, NaCl 0.2 percent and sterilized for 30min at 115 ℃.

The healthy laying hens comprise: no medicine or antibiotic is used within 3 months, and the energy provided by the concentrated feed accounts for less than or equal to 50 percent of the daily total energy requirement.

The specific treatment method of the scutellaria baicalensis leaves comprises the following steps: soaking stem and leaf of Scutellariae radix in 360L water for 1 hr, decocting for 50min, separating the liquid by hot press filtration, drying the residue at 85 deg.C for 30-45min, and concentrating the liquid to 5-10L.

The specific method for decocting the residue of the chicken-derived probiotic flora fermented scutellaria baicalensis stem and leaf with water comprises the following steps: mixing the water-decocted concentrated solution of the stem and leaf of the scutellaria with the dried and filter-pressed dregs of the stem and leaf of the scutellaria, and enabling the dregs to fully and uniformly absorb the liquid medicine to form a solid culture medium matrix of the stem and leaf dregs of the scutellaria;

inoculating 1% of the bacterial flora with high beta-glucosidase activity into a rich culture medium, keeping the temperature at 37 ℃, and carrying out shaking culture at 110 r/min for 12-48h to obtain a revived bacterial suspension; mixing 1-5L of revived bacterial suspension with 100Kg of Scutellaria baicalensis stem and leaf residue solid culture medium, and performing solid fermentation at normal temperature for 10-30 days.

Wherein: the formula of the rich culture medium is as follows: cellobiose 0.5%, streptomycin 0.02%, (NH4)2SO40.3%, MgSO4 & 7H2O 0.05%, KH2PO40.1%, NaCl 0.2%, sterilizing at 115 deg.C for 30 min;

the formula of the scutellaria baicalensis stem and leaf decoction dreg solid culture medium is as follows: 99Kg of culture medium matrix of scutellaria stem and leaf dregs solid, 400g of urea, (NH4)2SO 4400 g, 50g of MgSO4 & 7H2O 50 and KH2PO 4150 g.

And performing normal-temperature solid fermentation on the stem and leaf of the scutellaria baicalensis at the end of autumn in the harvesting period.

The concrete method for obtaining the poultry feed additive by molding comprises the following steps: drying the solid fermented crude scutellaria stem and leaf decoction dregs at 65 ℃ for 30-90min, then crushing the dregs into coarse powder without sieving, mixing 1kg of the crushed dregs with 1-3L of water decoction concentrated solution, then drying the mixture at 65 ℃ for 30-90min, crushing the mixture, sieving the dried mixture with a 40-mesh sieve, and sealing and packaging the crushed mixture to obtain the fermented crude scutellaria stem and leaf decoction poultry feed additive.

Through detection, the contents of total flavonoids, active isoflavones, digestible protein and short-chain fatty acid in the prepared poultry feed additive are obviously higher than those in the mixture of the pure scutellaria stem and leaf decoction and the dregs. The invention is beneficial to the high-efficiency resource utilization of the stem and leaf of the scutellaria baicalensis.

The invention utilizes chicken-derived probiotic flora with high beta-glucosidase activity to ferment the decoction dregs of the stem and leaves of the scutellaria baicalensis after being decocted in water, and the dried decoction dregs after being fermented are mixed with the concentrated water decoction solution to prepare the poultry feed additive through the processes of drying, crushing, granulating and the like, and the prepared feed additive has the contents of total flavonoids, active isoflavone, digestible protein and short-chain fatty acid which are obviously higher than the contents of the water decoction of the stem and leaves of the simple scutellaria baicalensis and the decoction dregs. Not only the dregs of decoction of the stem and leaves of the scutellaria baicalensis are fully utilized, but also the water decoction is organically fused with the dried fermented dregs of the decoction, thereby achieving the purpose of fully, efficiently and simply recycling the stem and leaves of the scutellaria baicalensis.

The fermented type scutellaria stem and leaf primary-decocted chicken feed additive utilizes chicken-derived high-beta-glucosidase activity flora to ferment dried scutellaria stem and leaf dregs by a solid fermentation method, the fermented dregs are also fused with water decoction concentrated solution of scutellaria stem and leaf, and finally the dried granular type feed additive is prepared, so that the feed additive is convenient to be mixed and fed in feed. The beta-glucosidase generated by the mixed bacteria not only decomposes and utilizes fiber substances in the medicinal residues of the stem leaves of the scutellaria baicalensis, but also can effectively convert flavonoid substances into an active form which can be directly absorbed by chickens, and the contents of total flavonoids, active isoflavones, digestible carbohydrates, digestible proteins and short-chain fatty acids in the formed feed additive can be obviously increased; the broiler feeding test result shows that the fermented scutellaria baicalensis stem and leaf primary-decocted chicken feed additive is added, so that 40-42-day-old and 62-64-day-old broilers can digest and utilize daily ration dry matter, crude protein and crude ash, and the apparent digestibility is obviously higher than that of an unfermented additive group. The feed additive for the original fried chicken with the fermented scutellaria baicalensis stem leaves has the beneficial effects of improving the production performance of broiler chickens and laying hens, increasing the disease resistance and the like.

Drawings

FIG. 1 is a preparation process diagram of the poultry feed additive of the invention.

Detailed Description

Materials and methods

1. Collecting and processing chicken and manure samples thereof: 10 healthy laying hens are raised scattered, any medicine and antibiotics are used within 3 months, and the energy provided by the concentrated feed accounts for no more than 50% of the total daily energy requirement. Weighing 5g of chicken manure sample, adding the chicken manure sample into sterilized 45mL of sterile water, keeping the temperature at 37 ℃, uniformly mixing the chicken manure sample by oscillation at 110 r/min, and centrifuging the chicken manure sample at 3000 r/min to obtain a bacterial suspension for later use.

2. Main liquid and medium:

gastric fluid (NaCl 2g, pepsin 3.2g, 36.5% HCl 7mL, sterile distilled water to volume 100 mL), simulated intestinal fluid (trypsin 0.9g, bile salts 6g, NaHCO 312.5 g, sterile distilled water dissolved and volume to 1000 mL).

Bran culture medium (bran 4.0%, urea 0.2%, (NH4)2SO 40.2%, MgSO 4.7H 2O 0.05%, KH2PO40.1%, sterilized at 115 ℃ for 30 min), enrichment medium (cellobiose 0.5%, streptomycin 0.02%, (NH4)2SO40.3%, MgSO 4.7H 2O 0.05.05%, KH2PO40.1%, NaCl 0.2%, sterilized at 115 ℃ for 30 min)

3. The simulated chicken gastrointestinal system is an in-situ continuous system, the first step is composed of gastric juice and bran culture medium, simulated small intestine liquid is added after the stomach simulation is finished, the pH value is adjusted to form a simulated small intestine environment, and the simulated small intestine environment enters a small intestine simulation stage in an original culture tube.

4. Pressure screening of chicken-derived high-beta-glucosidase activity mixed flora: 1mL of chicken dung bacteria suspension is put into a simulated chicken gastrointestinal system containing a bran culture medium and is subjected to shaking culture at the temperature of 37 ℃ and the speed of 110 r/min for 24 hours, 48 hours and 96 hours. After the culture is finished, 1mL of bacterial suspension is centrifugally taken in an enrichment medium at 3000 r/min, and the culture is carried out at 37 ℃ and 110 r/min in a shaking way.

5. Solid fermentation of scutellaria stem and leaf decoction dregs

A. Decocting and concentrating the stem and leaf of the scutellaria and processing dregs: 100Kg of stem and leaf of scutellaria baicalensis, 360 liters of water, soaking for 1 hour, and decocting for 50 minutes. Separating the medicinal liquid by hot press filtration, drying the residue at 85 deg.C for 30-45min, and concentrating the medicinal liquid to 5-10L.

B. Culture medium substrate and solid fermentation thereof: the scutellaria stem and leaf water decoction concentrated solution is mixed in the dried and filter-pressed scutellaria stem and leaf dregs to ensure that the dregs can fully and uniformly absorb the liquid medicine to form the scutellaria stem and leaf dregs solid culture medium matrix.

The preserved flora with high beta-glucosidase activity is inoculated in a rich culture medium by 1 percent of inoculum size, and is subjected to shaking culture at 37 ℃ and 110 r/min for 12-48 h.

Mixing 1-5L of revived bacterial suspension with 100Kg of scutellaria baicalensis stem and leaf decoction solid culture medium (99 Kg of scutellaria baicalensis stem and leaf decoction solid culture medium substrate, 400g of urea, (NH4)2SO 4400 g, 4.7H 2O 50g and KH2PO 4150 g), and carrying out solid fermentation at normal temperature (the harvesting period of scutellaria baicalensis stem and leaf is about the end of autumn) for 10-30 days.

6. Forming the fermented scutellaria baicalensis stem and leaf raw fried chicken feed additive: drying the solid fermented scutellaria baicalensis stem and leaf raw decoction dregs at 65 ℃ for 30-90 minutes, crushing, sieving with a 40-mesh sieve, sealing and packaging to finally obtain the fermented scutellaria baicalensis stem and leaf raw decoction chicken feed additive.

7. Analysis of beta-glucosidase activity: the DNS method is used for measuring the beta-glucosidase activity of the mixed flora.

And (3) preparing a crude enzyme solution. After the fermentation culture of the bran culture medium for 48 hours, the fermentation liquid is absorbed into a 1.5mL centrifuge tube and centrifuged at 8000 r/min, and the supernatant is the crude enzyme liquid.

Taking 0.5mL of crude enzyme diluted by 10-30 times into a colorimetric tube, adding 1mL of salicin solution (dissolved in 0.1M acetic acid buffer solution with pH 4.8), preserving the temperature for 30 minutes at 50 ℃, then adding 2 mL of DNS to stop the reaction, boiling for 10 minutes, adding distilled water after ice bath cooling to reach 12.5 mL, uniformly mixing, and measuring the OD value at 540 nm. The blank was treated with heat-inactivated enzyme in the same manner. The amount of enzyme required to produce 1. mu. mol of glucose from the substrate per minute was defined as one enzyme activity unit (IU).

8. Detecting active isoflavone by high performance liquid chromatography: the standard curve of soybean isoflavone is established by a mixed standard method, and the four standard products are daidzin, genistin, daidzein and genistein.

Accurately weighing 0.5 g of sample, dissolving in 80% methanol solution, ultrasonically oscillating for 30min, fixing the volume of 90% methanol solution to 50m L, centrifuging for 15 min at 10000 r/min, filtering supernatant with a filter membrane, and sucking 2 m L into a chromatographic bottle for later use. C18, 4.6X 150mm, 5 μm particle size stainless steel chromatographic column analysis of soy isoflavones.

9. Influence of fermentation type scutellaria baicalensis stem and leaf primary-fried chicken feed additive on broiler digestive metabolism

A. Animal grouping and rearing

Preparing a test daily ration: the daily ration used in the test is prepared according to the principle of equal energy and protein, and no antibiotic is added. Wherein 8% of dry mixture of unfermented radix Scutellariae stem and leaf crude decoction residue and decoction concentrate is added into control group (group I), and 8% of fermented radix Scutellariae stem and leaf crude decoction chicken feed additive is added into test group II.

Grouping and feeding: feeding 80 yellow-feathered broilers of 1 day age to a commercial ration for 14 days, after pre-feeding, weighing after fasting for 12 hours, selecting 60 healthy broilers according to the principles of similar body weight and halves of a male chicken and a female chicken, randomly dividing the healthy broilers into two groups, and feeding the two groups respectively with different feeds, namely a blank control group (group I) and a fermented scutellaria baicalensis stem-leaf raw fried chicken feed additive group (group II), wherein each group is repeated for 3 times, and each group is repeated for 10 chickens. The test chickens are fed according to free feeding and free drinking water and strictly according to the yellow-feathered broiler feeding management system.

B. Determination of apparent digestibility: and analyzing the apparent digestibility of the broiler chickens at the later growth stage (43-64 days old). During the last 3 days of each feeding stage, namely 40-42 days and 62-64 days, 100 g of chicken excrement is repeatedly collected every day, then the excrement collected for 3 days is repeatedly and uniformly mixed, the mixture is dried for 48 hours at the temperature of 65 ℃, and the mixture is crushed and stored at the temperature of-20 ℃ for analysis.

The apparent digestibility of the nutrients was determined using the endogenous indicator method, namely 4 mol/L hydrochloric Acid Insoluble Ash (AIA) referred to de Coca-Sinova et al (2011) method. The Dry Matter (DM), Crude Protein (CP) and crude Ash (Ash) contents of the feed and excrement were determined with reference to AOAC (1999), and the crude fat (EE) was determined by Soxhlet extraction.

Apparent digestibility of nutrient in ration (%) = 100- [ (content of certain nutrient in excreta%/content of certain nutrient in ration%) × (AIA content in ration/AIA content in excreta%) ] × 100.

10. Data processing and statistical analysis: data obtained by the test are sorted by using software Excel 2003, One-way ANOVA of SPSS16.0 statistical software is used for variance analysis, the LSD method is used for testing the difference significance among groups, the result is represented by the average value +/-standard deviation, and the judgment standard of the difference significance is P < 0.05.

Second, results and analysis

1. The content of active isoflavone and the conversion rate of aglycone of the fermented scutellaria baicalensis stem and leaf raw-fried chicken feed additive are as follows: the influence of fermentation of chicken-derived high beta-glucosidase activity mixed bacteria on the isoflavone content of the water-decocted scutellaria stem and leaf residues is shown in table 1, and the result shows that the content of three aglycones in the fermented scutellaria stem and leaf raw chicken feed additive is remarkably higher than the mixture of the water-decocted scutellaria stem and leaf residues and the concentrated solution.

TABLE 1 influence of fermentation of chicken-derived high beta-glucosidase activity mixed bacteria on isoflavone content in water-decocted Scutellariae radix stem and leaf residue (mg/kg)

FH, a fermented scutellaria baicalensis stem and leaf primary-fried chicken feed additive; WH, the mixture of the decoction dregs and the concentrated solution of the stem and leaf of the baical skullcap root; ab, different letters represent significant differences (P<0.05）。

The results of the conversion rate of aglycone are shown in table 2, and the results also show that the fermentation of the chicken-derived mixed bacteria with high beta-glucosidase activity can obviously convert isoflavone from a glucoside form into aglycone (table 2).

TABLE 2 influence of fermentation of chicken-derived high beta-glucosidase activity mixed bacteria on the conversion rate of water-decocted radix Scutellariae stem and leaf residue aglycone

FH, a fermented scutellaria baicalensis stem and leaf primary-fried chicken feed additive; WH, the mixture of the decoction dregs and the concentrated solution of the stem and leaf of the baical skullcap root; ab, different letters represent significant differences (P<0.05）。

2. Influence of fermentation type radix scutellariae stem and leaf primary-fried chicken feed additive on broiler digestibility

The research results are shown in table 3, which shows that the apparent digestibility of dry substances, crude proteins, crude fats and ash content of the broilers is obviously improved no matter the broilers are 40-42 days old or 62-64 days old by adding 8% of the fermented scutellaria baicalensis stem and leaf primary-decocted chicken feed additive into the daily feed of the broilers (the apparent digestibility of the dry substances, the crude proteins, the crude fats and the ash content of the broilers is obviously improved: (the feed additive isP<0.05) The contents of digestible protein, fat and carbohydrate can be increased by fermenting the mixed bacteria with high β -glucosidase activity to decoct the mixture of the stem and leaf dregs of the scutellaria baicalensis and the concentrated solution of the scutellaria baicalensis, the increase of the active isoflavone can also be one of the reasons, and the active isoflavone can promote the digestion and absorption of nutrient substances by regulating the immunity of the organism and the intestinal flora.

TABLE 3 influence of fermentation type Scutellaria baicalensis stem and leaf primary fried chicken feed additive on broiler digestibility (%)

FH, a fermented scutellaria baicalensis stem and leaf primary-fried chicken feed additive; WH, the mixture of the decoction dregs and the concentrated solution of the stem and leaf of the baical skullcap root; ab, different letters represent significant differences (P<0.05）。

The invention utilizes a mixed solid culture medium of stem and leaf dregs of scutellaria baicalensis and a water decoction concentrated solution after chicken-derived high beta-glucosidase activity flora fermentation and water decoction, the inoculation amount is 3 percent of bacterial suspension (prepared by the invention), and the fermentation is carried out at normal temperature before and after autumn equinox for 20 days. Compared with the mixed solid culture medium of the stem and leaf dregs of the scutellaria baicalensis after decoction and the decoction concentrated solution, the contents of total flavone, active isoflavone, digestible carbohydrate, digestible protein and short-chain fatty acid in the fermented scutellaria baicalensis stem and leaf primary-decocted chicken feed additive are obviously increased. The fermented scutellaria baicalensis stem and leaf primary-fried chicken feed additive can improve the production performance of broiler chickens and improve the economic benefit by improving the digestibility of dry matters, crude protein, crude fat and ash and increasing the content of active isoflavone in the feed.