CN112021467A - Application of fermented wormwood in preparation of products for improving production performance and oxidation resistance of broiler chickens - Google Patents
Application of fermented wormwood in preparation of products for improving production performance and oxidation resistance of broiler chickens Download PDFInfo
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- CN112021467A CN112021467A CN202010838482.3A CN202010838482A CN112021467A CN 112021467 A CN112021467 A CN 112021467A CN 202010838482 A CN202010838482 A CN 202010838482A CN 112021467 A CN112021467 A CN 112021467A
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Abstract
The invention discloses an application of fermented wormwood in preparing a product for improving the production performance and the oxidation resistance of broiler chickens, wherein the application of the fermented wormwood is as follows: preparing a product for reducing the diarrhea rate of the broiler chickens; preparing a product with a reduced material-to-weight ratio; preparing a product for improving digestibility of crude protein and crude fat; preparing a product for reducing the digestibility of crude protein in the feed; preparing a product for improving the tenderness and color of breast muscle and leg muscle of the broiler chicken; preparing a product with improved total oxidation resistance of the broiler chicken; preparing a product for enhancing the digestibility of the broiler chicken on the feed; preparing a product for reducing the depth of intestinal crypts of the broiler chickens; the product can improve the fluff arrangement condition of the broilers. The test of the invention shows that: the fermented wormwood rich in probiotics and antioxidant active ingredients is added into a broiler feed formula in a proper proportion, so that the broiler productivity can be improved, the broiler intestinal health and the organism antioxidant capacity are promoted, the meat quality is improved, and the broiler feed has high application value and market prospect.
Description
Technical Field
The invention relates to the technical field of biological feed preparation, in particular to application of fermented wormwood in preparation of a product for improving production performance and oxidation resistance of broiler chickens.
Background
The wormwood (Artemisia argyi) is a perennial herb of Artemisia of Compositae, is a traditional Chinese medicine in China, and has special medicinal and health-care values. The wormwood and the wormwood powder are also applied to animal production, and previous researches show that the addition of a certain amount of wormwood powder into the pig and chicken feed has the effects of promoting growth and health care, but the wormwood has high fiber content and special smell, and the digestion and utilization of monogastric animals are influenced due to overhigh feeding amount. The content of active antioxidant ingredients in the wormwood can be increased by the aid of the composite probiotics and the enzyme fermented wormwood, the wormwood is rich in probiotics, palatability of the wormwood is improved, and quality of the wormwood is improved, but application of the fermented wormwood in an animal feed formula and reports of improvement of broiler production performance and antioxidant capacity of the fermented wormwood are not available at home and abroad.
Disclosure of Invention
Therefore, the invention provides the application of the fermented wormwood in preparing products for improving the production performance and the oxidation resistance of broiler chickens.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an application of fermented wormwood in any one of the following:
(a1) preparing a product for reducing the diarrhea rate of the broiler chickens;
(a2) preparing a product with a reduced material-to-weight ratio;
(a3) preparing a product for improving digestibility of crude protein and crude fat;
(a4) preparing a product for reducing the digestibility of crude protein in the feed;
(a5) preparing a product for improving the tenderness and color of breast muscle and leg muscle of the broiler chicken;
(a5) preparing a product with improved total oxidation resistance of the broiler chicken;
(a6) preparing a product for enhancing the digestibility of the broiler chicken on the feed;
(a7) preparing a product for reducing the depth of intestinal crypts of the broiler chickens;
(a8) the product improves the arrangement condition of the chicken fluff;
(a9) a product for improving intestinal flora of broiler chicken.
The invention also provides the use of fermented wormwood in any one of the following:
(c1) preparing a product which reduces the content of malondialdehyde in the blood serum of the broiler chicken and improves the content of superoxide dismutase and glutathione peroxidase;
(c2) preparing a product which improves the enzyme activity of amylase, protease and cellulase in the digestive tract of the broiler chicken;
(c3) preparing a product for increasing the height of intestinal villi of the broiler chicken and the hiding ratio of the villi.
In one embodiment of the invention, the fermented wormwood is prepared by fermenting a fermentation liquor containing a wormwood fermentation medium with cellulase and composite probiotics, wherein the composite probiotics comprise lactobacillus casei, candida utilis and bacillus subtilis.
In one embodiment of the invention, the fermentation medium comprises the following raw materials in percentage by mass: 70% of wormwood, 24% of corn, 3% of wheat bran and 3% of glucose, adding 40% of distilled water, uniformly mixing the culture medium, and then preparing the product at 121 ℃ under high pressure for 20 min.
In one embodiment of the invention, the viable count of the lactobacillus casei, the candida utilis and the bacillus subtilis is 1.0 multiplied by 106CFU/mL、1.0×107CFU/mL and 1.0X 106CFU/mL。
The invention also provides any one of the following products:
(b1) preparing a product for reducing the diarrhea rate of the broiler chicken by using fermented wormwood;
(b2) preparing a product for reducing the material weight ratio by using fermented wormwood;
(b3) preparing a product for improving the digestibility of the crude protein and the crude fat by utilizing the fermented wormwood;
(b4) preparing a product for reducing the digestibility of crude protein in the feed by using fermented wormwood;
(b5) preparing a product for improving the tenderness and color of breast muscles and leg muscles of the broiler chicken by using fermented wormwood;
(b6) preparing a product with improved total oxidation resistance of the broiler chicken by using fermented wormwood;
(b7) preparing a product for enhancing the digestibility of the broiler chicken on the feed by utilizing the fermented wormwood;
(b8) preparing a product for reducing the depth of intestinal crypts of the broiler chicken by using fermented wormwood;
(b9) a product for improving the arrangement condition of the chicken fluff by utilizing fermented wormwood;
(b10) a product for improving intestinal flora of broiler chickens by using fermented wormwood.
The invention has the following advantages:
the test of the invention shows that: the fermented wormwood rich in probiotics and antioxidant active ingredients is added into a broiler feed formula in a proper proportion, so that the broiler productivity can be improved, the broiler intestinal health and the organism antioxidant capacity are promoted, the meat quality is improved, and the broiler feed has high application value and market prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
Fig. 1 is a section view of jejunum intestinal morphology of group a broilers provided by an embodiment of the present invention;
FIG. 2 is a section view of jejunum intestinal morphology of group B broilers provided by the embodiment of the present invention;
FIG. 3 is a diagram illustrating the situation that most of the E group broiler chickens jejunum villi are uniformly and orderly distributed in a columnar shape;
FIG. 4 is a composition diagram of species at the phylum of the jejunal content flora of broilers of different groups according to the embodiment of the present invention;
FIG. 5 is a composition diagram of species at a level of intestinal microbial flora of broilers of different groups according to an embodiment of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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.
Example 1 preparation of fermented Artemisia princeps Pampanini
1. Selecting crushed whole wormwood, crushing and sieving by a 60-mesh sieve. The probiotics is purchased from China general microbiological culture Collection center (CGMCC), Lactobacillus casei (Lactobacillus casei, strain number 1.2884) is cultured by MRS culture medium, Candida utilis (strain number 2.0615) is cultured by YPD culture medium, Bacillus subtilis (strain number 1.0504) is cultured by LB culture medium, the viable count of the three bacteria is respectively adjusted to 1.0 multiplied by 106CFU/mL、1.0×107CFU/mL and 1.0X 106CFU/mL, and mixing the three probiotics uniformly according to the equal volume ratio to prepare the composite probiotics. Cellulase (Cellulase) was purchased from Hippongze Biotech Ltd and the filter paper enzyme activity was 130 FPU/g.
The test adopts solid fermentation, and is divided into a control wormwood group and a fermented wormwood group, wherein the control group is not added with compound probiotics and enzyme, the test group is added with 1 percent of compound probiotics (v/w) and 1.5 percent of cellulase (w/w), and the test group is cultured for 4 days in a constant temperature box at 37 ℃.
2. Broiler formula with fermented wormwood feed
The feeding test was carried out at the schchang test base of the university of agriculture in the south of Henan, and 250 healthy AA broilers of 1d were selected and randomly divided into 5 treatment groups of 5 replicates each, each of which was 10 chickens. The feed is divided into two stages of feeding in the first stage (1-21d) and feeding in the later stage (22-42d), and the feed formula and nutritional ingredients of the fermented wormwood added in the two stages are shown in tables 1 and 2. Adopting multi-layer cage culture, freely feeding, naturally ventilating, and artificially illuminating (after 7d, 23h illumination, 1h darkness); the immunization program is as follows: 7d of new-branch combined vaccine and 21d of new-branch combined vaccine.
The experimental groups were as follows: group A: basal ration; group B: basal ration + 3% unfermented wormwood; group C: basal diet + 1.5% fermented wormwood; group D: basal diet + 3.0% fermented wormwood; group E: basic ration plus 4.5% fermented wormwood
TABLE 1 composition and nutritional level (%, air-dried base) of broiler early stage (0-21d) daily ration
Note: 1. the vitamins and minerals provided by each kilogram of feed comprise 100mg of iron (ferrous sulfate); zinc (zinc oxide) 60 mg; manganese (manganese oxide) 80 mg; copper (copper sulfate) 8 mg; iodine (potassium iodate) 0.45 mg; selenium (sodium selenite) 0.35 mg; vitamin A12000 IU; vitamin D33000 IU; vitamin E20 IU; vitamin K31 mg; 35mg of nicotinic acid; 10mg of pantothenic acid; 6.0mg of riboflavin; vitamin B120.01 mg; biotin 0.15 mg; folic acid 1.25 mg; pyridoxine 3.5 mg; thiamine 2.0 mg.
TABLE 2 composition and nutritional level (%, air-dried basis) of broiler early stage (22-42d) daily ration
Note: as in table 1.
Example 2 production Properties
1. The weight was measured at 1d, 21d, 42d, respectively, and the amount of food intake was recorded every day for each repetition. Diarrhea, death status was recorded for each replicate chicken. Average Daily Feed Intake (ADFI) and Average Daily Gain (ADG) were calculated. And calculating the feed conversion rate and the diarrhea rate, wherein the diarrhea rate is total diarrhea days/(total number of chicken multiplied by test days).
2. As can be seen from Table 3, in the early stage of broiler test, the diarrhea rate of broilers is reduced by adding wormwood and fermenting wormwood to the broiler feed, and the group D with 3% fermented wormwood has the optimal effect.
TABLE 3 influence of different treatment wormwood feed formulations on growth performance of 1-21d broilers (g)
Note: the data in the same column are marked with different lower case letters to indicate significant difference (P <0.05), and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 4, the addition of 3% fermented wormwood (group D) significantly increased the daily gain (P <0.05) in the later stage of broiler chickens, and decreased the feed-weight ratio and diarrhea rate (P < 0.05). The feed-weight ratio and diarrhea rate of the later-period broiler chickens are also reduced by other groups added with wormwood (P is less than 0.05).
Table 4. influence of different treatment wormwood feed formulas on growth performance of 22-42d broilers
Example 3 determination of nutrient digestibility
1. The excrement is collected at 19-21d and 40-42d respectively by adopting a total excrement collecting method (the feed intake and the excretion are recorded for 3 days). Mixing feces samples every day to remove impurities, adding 10% hydrochloric acid to fix nitrogen, freezing and storing in a refrigerator at-20 ℃, mixing the feces samples for three days after the test is finished, drying at 65 ℃, fully dampening and crushing, and then determining various nutritional indexes: crude Protein (CP) is prepared from national standard CB/T6432-94; crude fat (EE) is prepared by adopting national standard GB/T6433-2006; calcium (Ca) is prepared by complexometric titration of disodium edetate; phosphorus (P): the national standard GB/T6437-2002 is adopted.
2. Influence of bacterium and enzyme combined treatment of wormwood on nutrient digestibility of broiler chickens
As can be seen from table 5, the addition of 3% fermented wormwood (group D) to the feed significantly increased the digestibility of crude protein and crude fat (P <0.05) at the early stage of broiler feeding.
Table 5 influence of different treatment wormwood feed formulas on nutrient digestibility (%)
As can be seen from Table 6, the addition of 3% of fermented wormwood (group D) in the feed significantly improves the digestibility of crude fat and calcium in the later period of broiler chickens, while the addition of 4.5% of fermented wormwood significantly reduces the digestibility of crude protein in the feed (P < 0.05).
TABLE 6 influence of different treatments of mugwort on digestibility of nutrients in broiler chickens 40-42d (%)
Example 4 measurement of slaughtering Performance of meat chickens
1. At 42d, one chicken was taken per replicate and meat quality measurements were performed after slaughter.
Acidity: and measuring the pH values of the slaughtered pectoral muscles and leg muscles within 45min and after 24h by using a testo 205 pH/acidity measuring instrument.
Muscle drip loss: weighing two 10g of chest muscles and leg muscles respectively for each chicken (W1), placing in a dripping water loss measuring tube to avoid adherence, taking out after 24h in a refrigerator at 4 ℃, wiping off water on the surface of the muscles by using filter paper and weighing (W2), and calculating the dripping water loss, wherein the dripping water loss is (W1-W2)/W1 multiplied by 100 percent)
And (3) meat color determination: after slaughter, the meat sample was trimmed to a circular shape having a thickness of about 0.5cm, and the trimmed meat sample was placed in a CHROMA METER CR-400 colorimeter to measure the L (light), a (red), and b (blue) values of the pectoral muscles, and each of the pectoral muscles and the leg muscles was measured 2 times, and the average value was taken.
And (3) cooked meat rate determination: after the chest muscles and the leg muscles are aged in a refrigerator at 4 ℃ for 24h, the muscles to be measured are weighed (W1), the muscles are boiled in a water bath at 100 ℃ for 30min, taken out and hung in a shade for 20min, and the muscles are wiped by filter paper to dry the surface water and weighed (W2).
Cooked meat rate W2/W1 × 100%
And (3) shear force measurement: putting a fresh meat sample into a refrigerator with the temperature of 4 ℃ for 48h for curing, taking out the meat sample, standing the meat sample at room temperature for 1h until the temperature of the meat sample is raised to room temperature, inserting a thermometer into the central part of the meat sample, then putting the meat sample into a thermostatic water bath with the temperature of 80 ℃ for heating until the central temperature of the meat sample reaches 70 ℃, stopping heating, taking out the meat sample when the meat sample is cooled to about 20 ℃, cutting the pectoral muscle into strips with the length of 3 multiplied by 2 multiplied by 1cm along the direction of muscle fibers of the meat sample, cutting the leg muscles into strips with the length of 2 multiplied by 1 multiplied by 0.5cm, putting the meat sample on a C-LM 3B digital display type muscle tenderness instrument for cutting at room temperature, and.
2. Influence of different treatment of wormwood feed on meat quality of broiler chickens
Important indexes for evaluating the meat quality of poultry mainly comprise pH value, water binding capacity, shearing force, meat color and the like. The pH value of the poultry muscle is divided into pH 45min and pH 12 h. The poultry muscular system hydraulic power evaluation has direct measurement and also has drip loss, cooking loss, moisture, cooked meat rate and the like, while the shearing force is an important index of the tenderness of poultry muscles, and the smaller the shearing force is, the more tender the meat quality is. The color value of muscle color is divided into three indexes of a value, L value and b value, wherein the a value represents red and green, the L value represents brightness (black and white), and the b value represents yellow and blue. a higher value of a indicates a higher myoglobin content in the muscle. As can be seen from tables 7 and 8, the feed formula with 3% of fermented wormwood is added remarkably improves the a value of the breast muscle of the broiler chicken and improves the meat color. The formula of the fermented wormwood feed added with 4.5% has better effect of improving the tenderness and color of breast muscle and leg muscle of the broiler chicken.
Table 7 effect of different treatment wormwood feed formulations on breast muscle meat quality (%)
Table 8 effect of different treatment wormwood feed formulations on leg muscle quality (%)
Example 5 measurement of Biochemical index and antioxidant index of broiler serum
1. At 42d, 1 chicken was randomly selected per replicate, carotid blood was taken, 5mL each, centrifuged at 3000rpm for 10min, and serum was isolated. The biochemical indexes of each serum are measured by a blood biochemical semi-automatic analyzer.
The determination of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC) and Malondialdehyde (MDA) in serum is carried out by adopting a kit produced by Nanjing institute of bioengineering.
2. Influence of different treatments of wormwood on oxidation resistance of broiler chickens
From table 9, it can be known that the addition of 3% (group D) and 4.5% (group E) fermented wormwood significantly reduced the malondialdehyde content in the chicken serum, significantly increased the contents of superoxide dismutase and glutathione peroxidase, and increased the total antioxidant capacity of broiler chickens (P < 0.05).
TABLE 9 influence of different treated wormwood feed formulas on broiler serum antioxidant index
Example 6 determination of digestive enzyme Activity in intestinal tract of broiler chickens
1. At 42 days, randomly selecting 1 chicken per repetition for slaughtering, placing the small intestine content in a 2mL sterilized freezing tube, rapidly transferring into liquid nitrogen, and storing at-80 deg.C.
(1) The protease activity is determined according to the neutral protease activity determination method recommended by national standard (1999), namely the Folin method.
1) Reagent and preparation: phosphate buffered saline PBS preparation: 8g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate and 0.2g of dipotassium hydrogen phosphate are dissolved in 900mL of distilled water, and then concentrated hydrochloric acid is adjusted to pH 7.2 to achieve a constant volume of 1L. After passing through a 0.22 μm filter, the mixture was stored at room temperature.
Preparation of 1% casein: 0.5g (to the nearest 0.001) of casein, 0.1mol/L of sodium hydroxide 5mL was added, and after dissolution under heating, the volume was adjusted to 100mL with PBS, and the mixture was stored at 4 ℃.
Preparation of tyrosine standard solution: drying tyrosine to constant weight at 65 ℃, adding 6mL of 1mol/L hydrochloric acid into the tyrosine with the accuracy of 0.1g, dissolving, and then using 0.2mol/L hydrochloric acid to fix the volume to 100mL, wherein the concentration is 1000 mu g/mL, taking 1mL out, using 0.2mol/L hydrochloric acid to fix the volume to 10mL, namely 100 mu g/mL.
Preparation of 0.4M sodium carbonate: 21.2g of anhydrous sodium carbonate is taken, and dissolved by distilled water to reach the constant volume of 500 mL.
Preparation of 0.4M trichloroacetic acid: 65.4g of trichloroacetic acid is taken, and the trichloroacetic acid is dissolved in distilled water to be constant volume to 1L.
Folin phenol: a Chinese medicine group.
2) Preparing a standard curve, preparing tyrosine solutions (0, 10, 20, 30, 40, 50 and 60mg/mL) with different concentrations, taking 7 test tubes, respectively adding 1mL of the tyrosine solutions with different concentrations, then adding 5mL of a sodium carbonate solution with the concentration of 0.4M and 1mL of a Folin working solution, preserving the temperature at 40 ℃ for 20min, and measuring the absorbance at the wavelength of 660 nm.
3) And (3) measuring the activity of the sample protease, accurately weighing 0.5g of intestinal contents, adding 4.5mL of PBS buffer solution into a 10mL centrifugal tube, performing vortex oscillation for 1h, standing, and taking supernate as a sample diluent. Taking 1mL of sample diluent, heating at 40 ℃ for 2min, adding 1mL of 1% casein preheated at 40 ℃, accurately preserving heat for 10min, immediately adding 2mL of trichloroacetic acid with the concentration of 0.4M to terminate the reaction, continuing preserving heat for 20min, centrifuging at 8000r/min for 5min, taking 1mL of supernatant in a new test tube, adding 5mL of sodium carbonate solution with the concentration of 0.4M and 1mL of Folin working solution, carrying out warm water bath at 40 ℃ for 20min, measuring the absorbance at the wavelength of 660nm, and preparing a blank control sample: 0.4M trichloroacetic acid was added prior to casein addition and the other operations were as above.
4) Protease activity calculation, 1mg tyrosine produced per minute at 40 ℃ was defined as 1 protease activity unit (U). Determination of tyrosine content/reaction time × 4 × dilution times of sample/sample weight
(2) Determination of amylase activity:
1) reagent and preparation, phosphate buffer PBS preparation: 8g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate and 0.2g of dipotassium hydrogen phosphate are dissolved in 900mL of distilled water, and then concentrated hydrochloric acid is adjusted to pH 7.2 to achieve a constant volume of 1L. After passing through a 0.22 μm filter, the mixture was stored at room temperature.
Preparation of 1% casein: 0.5g (to the nearest 0.001) of casein, 0.1mol/L of sodium hydroxide 5mL was added, and after dissolution under heating, the volume was adjusted to 100mL with PBS, and the mixture was stored at 4 ℃.
Preparation of DNS: 3.15g of 3, 5-dinitrosalicylic acid is put into a 1000mL beaker, 500mL of distilled water is added, after the solution is stirred and dissolved in a water bath at 45 ℃, 100mL of 0.2g/mL of sodium hydroxide is added, the solution is stirred once every 1min until the solution is clear, 91g of potassium sodium tartrate, 2.5g of phenol and 2.5g of anhydrous sodium sulfite are continuously and slowly added, 300mL of distilled water is supplemented, the solution is stirred in the water bath until no solid matter is seen, the volume is determined to be 100mL after cooling, the solution is filtered by filter paper, and the solution is stored at room temperature in a dark place.
Preparation of 1% starch solution: weighing 1g of starch solution, putting the starch solution into a beaker, adding 5mL of distilled water for wetting, putting the beaker into an electric furnace, adding 95mL of boiling water, heating the beaker again to boil, cooling the beaker for use, and using the beaker after the beaker is cooled.
2) And (3) measuring the sample, accurately weighing 0.5g of intestinal tract content sample, adding 4.5mL of PBS buffer solution, carrying out vortex oscillation for 1h, standing, and taking supernatant as sample diluent. Adding 1mL of sample diluent into a 25mL graduated tube, carrying out water bath at 40 ℃ for 10min, adding 1mL of starch solution, uniformly mixing by shaking, carrying out water bath at 40 ℃ for 10min, respectively adding 3mL of DNS (domain name system), boiling for 5min, cooling to room temperature, fixing the volume to 25mL, and taking the measured OD value at 540nm as the measured OD value of the sample; adding 1mL of sample diluent into a 25mL graduated tube, respectively adding 3mL of DNS, uniformly shaking, carrying out water bath at 40 ℃ for 10min, adding 1mL of starch solution, uniformly shaking, carrying out water bath at 40 ℃ for 10min, boiling for 5min, cooling to room temperature, fixing the volume to 25mL, and measuring an OD value at 540nm as a blank OD value of the sample; the sample diluent was replaced with distilled water, DNS addition was performed in the same manner as the sample blank determination, no starch solution was added, and the standard blank OD value was measured at 540 nm.
3) Calculating the activity of amylase, wherein the content of reducing sugar in the sample is the content of the sample determined, namely the blank content of the sample; the enzyme amount for producing 1mg of glucose in 1min is one enzyme activity unit (u); amylase activity (u/g) is equal to the content of reducing sugar in the sample, the total volume of the sample diluent, the dilution factor, the weight of the sample, the volume of enzyme used for determination and the like, and the concentration of the enzyme is 5min
(3) Determination of cellulase activity:
1) reagent preparation, 0.2mol/L acetic acid solution: dissolving 12mL of glacial acetic acid in distilled water, and fixing the volume to 1L. 0.2mol/L sodium acetate solution: 27.2g of crystalline sodium acetate was weighed out and dissolved in distilled water to a constant volume of 1L. 0.2mol/L acetate-sodium acetate buffer (pH 4.5): a certain amount of 0.2mol/L acetic acid solution and 0.2mol/L sodium acetate solution are taken to be combined until the pH value is 4.5.
Preparation of standard DNS reagents: 3.15g of 3, 5-dinitrosalicylic acid is put into a 1000mL beaker, 500mL of distilled water is added, after the solution is stirred and dissolved in a water bath at 45 ℃, 100mL of 0.2g/mL of sodium hydroxide is added, the solution is stirred once every 1min until the solution is clear, 91g of potassium sodium tartrate, 2.5g of phenol and 2.5g of anhydrous sodium sulfite are continuously and slowly added, 300mL of distilled water is supplemented, the solution is stirred in the water bath until no solid matter is seen, the volume is determined to be 100mL after cooling, the solution is filtered by filter paper, and the solution is stored at room temperature in a dark place.
Glucose standard stock solution (10 mg/mL): 1g of glucose dried at 105 ℃ is taken to be accurate to 0.1mg, dissolved by acetic acid-sodium acetate solution and fixed to 100 mL.
Glucose standard use solution: taking 0.00 mL, 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, 5.00 mL, 6.00 mL and 7.00mL of the glucose standard stock solution respectively, adding acetic acid-sodium acetate solution to a constant volume, and shaking up for later use.
2) And (3) preparing a standard curve, namely adding 2mL of glucose standard use solution with different concentrations into a 25mL graduated tube, adding 2mL of sodium acetate buffer solution, adding 5mL of DNS respectively, shaking for 1min, uniformly mixing, boiling for 5min, cooling to room temperature, fixing the volume to 25mL, using distilled water to replace glucose standard solution as a blank control for zero setting, and measuring an OD value at 540 nm. And drawing a standard curve by taking the glucose content as an abscissa and the absorbance as an ordinate.
3) And (3) measuring the sample, accurately weighing 0.5g of intestinal tract content sample, adding 4.5mL of physiological saline, shaking for 1h in a vortex, centrifuging for 5min at 10000r/min, and taking the supernatant as a crude enzyme solution. Placing a (1cm multiplied by 6cm) filter paper strip at the bottom of a 25mL graduated tube, accurately adding 2mL of corresponding pH 4.5 acetic acid-sodium acetate buffer solution, soaking the filter paper strip by the solution in the test tube, placing 2mL of diluted crude enzyme solution in the test tube (replacing the crude enzyme solution with distilled water in blank control), shaking, uniformly mixing, covering and plugging, placing in a 50 ℃ water bath kettle for 60min, adding 5mL of DNS reagent into each tube, carrying out boiling water bath for 5min, cooling to room temperature, and fixing the volume of distilled water to 540nm of a spectrophotometer with 25mL to measure the absorbance.
4) And (4) calculating the activity of the cellulase (u/g), wherein the activity of the cellulase (u/g) is the content of reducing sugar in the sample, the total volume of the dilution solution of the sample, the dilution times/weight of the sample/reaction time.
2. Influence of different treatments of wormwood on digestive enzyme activity and morphology of broiler intestinal tract
As can be seen from Table 10, the 3% addition of the fermented wormwood feed group (group D) significantly improves the enzyme activities of amylase, protease and cellulase in the digestive tract of the broiler chickens (P is less than 0.05), and enhances the digestibility of the broiler chickens on the feed.
TABLE 10 influence of different treated wormwood feed formulas on intestinal enzyme activity (U/g) of broilers
Example 7 determination of the height of villi in the Small intestine of broiler chickens and the depth of crypts
1. At 42 days, 1 chicken was randomly selected per repetition for slaughter, and the jejunum tube was taken about 2cm and fixed with 10% formaldehyde. Sending to Zhengzhou university basic medical college for detection. After washing, correcting, washing, dehydrating, transparentizing and embedding, paraffin sections are manufactured, and the intestinal villus length and the crypt depth are measured through HE dyeing, xylene transparentizing treatment, mounting microscopic examination after neutral resin is dripped on the edges of the sections.
Immunohistochemistry step (Sp method): dewaxing the slices to water, and washing for 3-5min by PBS; repairing citric acid antigen for 10min, and naturally cooling; soaking in PBS for 5min, 3 times (3 × 5 min); blocking endogenous peroxidase with 3% H2O2, soaking in PBS for 5min at room temperature for 3 times (3 × 5min) at 20 min; dripping 50 μ L goat serum per tablet, and sealing endogenous biotin at room temperature for 20 min; throwing off and washing, dripping 50 mu L primary antibody (1: 100) per tablet, and standing overnight at 4 ℃; soaking in PBS for 5min, 3 times (3 × 5 min). Dripping 50 mu L of second antibody per tablet at 37 ℃ for 30 min; soaking in PBS for 5min, 3 times (3 × 5 min); dripping 50 mu L/piece of horseradish enzyme labeled oomycin leucosin at 37 ℃ for 30 min; soaking in PBS for 5min, 3 times (3 × 5 min); DAB color development, microscopic control and tap water suspension; performing hematoxylin counterstaining and performing gradient alcohol dehydration; xylene transparent, neutral gum mounting.
2. Influence of different treatments of wormwood on intestinal morphology of broiler chickens
Table 11 shows that the formula of the feed in group D with 3% fermented wormwood significantly increases the height of intestinal villi and the hiding ratio of villi in broiler chicken (P <0.05), and decreases the depth of intestinal crypts (P < 0.05).
In the intestinal morphology, the arrangement of the chicken jejunum villi in the group A (figure 1) is irregular, the chicken jejunum villi in the group B (figure 2) and the chicken jejunum in the group E (figure 3) which are added with 3 percent of normal wormwood and 3 percent of bacteria enzyme for combined treatment of wormwood are mostly uniformly and orderly distributed in a columnar shape, and the arrangement condition of the villi is effectively improved. The formula of the fermented wormwood feed added with 3% is more beneficial to the intestinal health of the broiler chicken.
TABLE 11 influence of different treatments of wormwood feed on the jejunum villus height and crypt depth of broilers
Example 8 determination of intestinal microbial flora of broiler chickens
1. At 42 days, randomly selecting 1 chicken per time, slaughtering, storing jejunum content in 2mL freezing tube with liquid nitrogen, and transferring to-80 deg.C refrigerator. Jejunum content samples were sent to the Shanghai Meiji BioLimited for determination and analysis of microbial diversity.
The sample sequencing procedure was as follows: sequencing experiment process: extracting sample DNA; designing a synthetic primer joint; PCR amplification and product purification; quantifying and homogenizing a PCR product; constructing a PE library; miseq sequencing.
DNA extraction: after the extraction of the group ONA, the extracted genomic DNA was detected using 1% agarose cryo-ice.
And (3) PCR amplification: specific primers with barcode were synthesized according to the designated sequencing region.
In order to ensure the accuracy and reliability of the post-allocation data extension, two conditions need to be met, 1) label-based recycling and amplification are used as far as possible. 2) Ensure the amplification cycle of each sample. Representative samples were randomly selected for pre-experiments to ensure that the majority of samples were able to amplify the correct concentration of the contents at the lowest cycle number. For PCR, TransGen AP221-02 and TransStart Fastplu DNA Polymerase were used. A PCR instrument: ABI
Model 9700.
All samples were run under the standard experimental conditions, 3 replicates per sample, PCR products from the same sample were mixed and detected by 2% agarose gel electrophoresis, PCR products were recovered by cutting with AxyPrepDNA gel recovery kit (AXYGEN Co.), eluted with Tris-HCl, and detected by 2% agarose electrophoresis.
Fluorescence quantification: referring to the preliminary quantitative result of electrophoresis, the PCR product is mixed by a quantiFluor TM-ST blue fluorescence quantitative system according to the corresponding proportion according to the sequencing quantity requirement of each sample.
Construction of Miseq library: 1) adding a luminal adaptor sequence to the outer end of the target region by PCR; 2) cutting gel by using a gel recovery kit to recover a PCR product; 3) eluting with Tris-HCL buffer solution, and detecting by 2% agarose electrophoresis; 4) sodium hydroxide denaturation produces single-stranded DNA fragments. Reagent: TruSeqTM DNA Sample Prep Kit.
Miseq sequencing: one end of the DNA fragment is complementary with the basic group of the primer and trapped on the chip; using the DNA fragment as a template and a base sequence fixed on the chip as a primer to carry out PCR synthesis, and synthesizing a target DNA fragment to be detected on the chip; after denaturation and fire, the other end of the DNA fragment on the chip is complementary with another primer nearby and is also fixed to form a bridge; performing PCR amplification to generate a DNA cluster; the DNA amplicon is linearized into a single strand; adding modified DNA polymerase and 4 kinds of fluorescence labeled dNTPs, and synthesizing only one base in each cycle; scanning the surface of the reaction plate by laser, and reading the nucleotide species polymerized by the first round of reaction of each template sequence; chemically cutting the fluorescent group and the terminating group, recovering the 3' end viscosity, and continuing to polymerize a second nucleotide; and counting the fluorescent signal results collected in each round to obtain the sequence of the template DNA.
3. And (3) data analysis: the test data were analyzed using SPSS 17.0 statistical software and multiple comparisons of groups were performed using the Duncan method, with P <0.05 being significantly different and the results being expressed as mean ± standard deviation.
The intestinal microflora statistical method adopts the mothur to calculate the diversity index and the species relative abundance, uses an R language tool to make a community bar chart and a curve chart, and uses One-way ANOVE to analyze the difference of sample components.
2. Effect of different treatments of Artemisia argyi on intestinal microflora of broilers
And (3) analyzing the abundance and diversity of the intestinal content flora of broilers of different groups: from the analysis of the abundance and diversity of intestinal flora of broilers (table 12), it can be seen that significant differences exist between the Shannon index and Simpson index in the group B of intestinal flora to which 3% of the non-fermented wormwood is added and the group a of the control group, which indicates that the feed formula to which the non-fermented wormwood is added significantly affects the abundance of microbial flora (P <0.05), while the group E index and the control group to which the 3% of the fermented wormwood is added do not significantly differ, indicating that the feed formula to which the wormwood is fermented helps to maintain the balance of intestinal microbial flora of broilers. The coverage rate is an important index for evaluating the sequencing condition of the intestinal microflora, the coverage rate of the sequencing sample of the test is over 99.7 percent, the sample is fully sampled, the sequencing is reasonable, and the sequencing result can truly reflect the composition condition of each group of intestinal microflora.
TABLE 12 analysis of enrichment and diversity of jejunum content flora in various treated wormwood feed groups
Analyzing species composition of jejunum content flora of broilers of different groups: as can be seen from FIG. 4 and Table 13, the intestinal microflora of broiler chickens mainly consists of the phylum Firmiutes Firmicutes, Actinobacteria actinomycetes, Cyanobactria Cyanobacteria, Bacteroides Bacteroidetes and Proteobacteria Proteobacteria. Firmicutes is the dominant phylum in the intestinal tracts of broiler chickens, and studies show that Firmicutes enzyme can promote the metabolism and utilization of energy. The 3% wormwood group (group B) and the control group were added to significantly reduce the firmicutes, while the number of other phyla was significantly increased, which disturbed the balance of intestinal flora at phylum level, while group E (with 3% fermented wormwood) helped balance of intestinal flora by reducing the effect of wormwood addition alone.
Table 13 horizontal species composition (%) -of intestinal flora of broilers of different treatment groups
As can be seen from fig. 5 and table 14, the species of the broiler intestinal microbial flora in the species level was significantly increased in the 3% wormwood addition group (group B) and the control group (group a) compared to the dominant microbial flora, and the microbial flora was significantly changed. Compared with the control group, the group added with the fermented wormwood (group D) remarkably increases the number of beneficial bacteria such as Lactobacillus (P <0.05) and reduces the number of bacillus (Turcibacter) (P < 0.05); compared with the group B, the number of mouse rhinoceros (Nasimurium), Lactobacillus salivarius (Lactobacillus _ salivaria), Streptococcus (Streptococcus _ pluralium), Streptococcus (Streptococcus), and Phaseolus vulgaris (Phaseolus _ vulgaris) is remarkably reduced (P <0.05), the number of Lactobacillus (Lactobacillus) and Turcibacter (Turcibacter) is remarkably increased (P <0.05), and the balance of intestinal flora is remarkably improved.
TABLE 14 broiler chicken intestinal flora level species composition (%)
The results show that the broiler feed added with 3% of fermented wormwood remarkably improves the production performance and feed nutrient digestibility of broilers, promotes the balance of intestinal structures and microbial flora, improves the muscle quality of broilers, and enhances the oxidation resistance of broilers.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. Use of fermented wormwood in any one of:
(a1) preparing a product for reducing the diarrhea rate of the broiler chickens;
(a2) preparing a product with a reduced material-to-weight ratio;
(a3) preparing a product for improving digestibility of crude protein and crude fat;
(a4) preparing a product for reducing the digestibility of crude protein in the feed;
(a5) preparing a product for improving the tenderness and color of breast muscle and leg muscle of the broiler chicken;
(a5) preparing a product with improved total oxidation resistance of the broiler chicken;
(a6) preparing a product for enhancing the digestibility of the broiler chicken on the feed;
(a7) preparing a product for reducing the depth of intestinal crypts of the broiler chickens;
(a8) the product improves the arrangement condition of the chicken fluff;
(a9) a product for improving intestinal flora of broiler chicken.
2. Use of fermented wormwood in any one of:
(c1) preparing a product which reduces the content of malondialdehyde in the blood serum of the broiler chicken and improves the content of superoxide dismutase and glutathione peroxidase;
(c2) preparing a product which improves the enzyme activity of amylase, protease and cellulase in the digestive tract of the broiler chicken;
(c3) preparing a product for increasing the height of intestinal villi of the broiler chicken and the hiding ratio of the villi.
3. The use according to claim 1,
the fermented wormwood is prepared by fermenting a fermentation liquid containing a wormwood fermentation culture medium with cellulase and composite probiotics, wherein the composite probiotics comprise lactobacillus casei, candida utilis and bacillus subtilis.
4. The use according to claim 3,
the fermentation medium comprises the following raw materials in percentage by mass: 70% of wormwood, 24% of corn, 3% of wheat bran and 3% of glucose, adding 40% of distilled water, uniformly mixing the culture medium, and then preparing the product at 121 ℃ under high pressure for 20 min.
5. The use according to claim 3,
the viable count of the lactobacillus casei, the candida utilis and the bacillus subtilis is 1.0 multiplied by 106CFU/mL、1.0×107CFU/mL and 1.0X 106CFU/mL。
6. Any one of the following products:
(b1) preparing a product for reducing the diarrhea rate of the broiler chicken by using fermented wormwood;
(b2) preparing a product for reducing the material weight ratio by using fermented wormwood;
(b3) preparing a product for improving the digestibility of the crude protein and the crude fat by utilizing the fermented wormwood;
(b4) preparing a product for reducing the digestibility of crude protein in the feed by using fermented wormwood;
(b5) preparing a product for improving the tenderness and color of breast muscles and leg muscles of the broiler chicken by using fermented wormwood;
(b6) preparing a product with improved total oxidation resistance of the broiler chicken by using fermented wormwood;
(b7) preparing a product for enhancing the digestibility of the broiler chicken on the feed by utilizing the fermented wormwood;
(b8) preparing a product for reducing the depth of intestinal crypts of the broiler chicken by using fermented wormwood;
(b9) a product for improving the arrangement condition of the chicken fluff by utilizing fermented wormwood;
(b10) a product for improving intestinal flora of broiler chickens by using fermented wormwood.
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Application publication date: 20201204 |