CN117378702A - Poultry feed additive and application thereof - Google Patents
Poultry feed additive and application thereof Download PDFInfo
- Publication number
- CN117378702A CN117378702A CN202311228608.5A CN202311228608A CN117378702A CN 117378702 A CN117378702 A CN 117378702A CN 202311228608 A CN202311228608 A CN 202311228608A CN 117378702 A CN117378702 A CN 117378702A
- Authority
- CN
- China
- Prior art keywords
- feed additive
- lactobacillus brevis
- poultry
- fermentation
- feed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
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Abstract
The invention relates to the field of poultry raising, in particular to a poultry feed additive and application thereof. The invention aims to solve the technical problem of providing an avian feed additive and application thereof, which can improve the growth performance of birds, enhance the immunity of organisms and improve the antioxidation function. The poultry feed additive is used for fermenting poultry daily ration, the fermented daily ration can improve the growth performance of poultry, enhance the immunity of organisms, improve the antioxidation function, improve intestinal villus structure, increase the number of mucous membrane goblet cells, increase the absorption area of intestinal mucous membranes and promote the absorption of nutrient substances by small intestines.
Description
Technical Field
The invention relates to the field of poultry raising, in particular to a poultry feed additive and application thereof.
Background
The microbial fermented feed is prepared by adding a certain proportion of microorganisms and moisture into daily ration, uniformly mixing, and then carrying out aerobic and anaerobic fermentation at a certain temperature. In recent years, along with the continuous progress of microbial application research at home and abroad, the application of preparing biological feed by utilizing beneficial microbial fermentation in livestock and poultry cultivation is increasingly paid attention to.
It is reported that the daily ration of livestock and poultry can produce more organic acid and release more micromolecular substances through microbial fermentation, and has obvious effects of maintaining the balance of gastrointestinal tracts of livestock and poultry, improving the feed conversion rate and enhancing the immunity of organisms. The metabolite produced after fermentation has various biological activities such as antibacterial, antioxidant, mildew toxin degrading, anti-nutritional factor decomposing, etc.
In recent years, the harm caused by the abuse of antibiotics in animal husbandry breeding is more and more serious, and the rising of the price of raw materials in recent two years is added, so that the cost of poultry breeding is increased sharply, and the healthy development of the poultry breeding is affected. In order to reduce the use of antibiotics, improve the conversion efficiency of feed, reduce the cost of poultry farming and ensure the healthy and stable development of poultry farming. It is necessary to deeply study the influence of the microbial fermentation feed on the production performance of the poultry, and a foundation is laid for the application of the microbial fermentation feed in the poultry cultivation.
Disclosure of Invention
The invention aims to solve the technical problem of providing an avian feed additive and application thereof, which can improve the growth performance of birds, enhance the immunity of organisms and improve the antioxidation function.
The invention is realized in the following way:
the poultry feed additive comprises a composite bacterium, wherein the composite bacterium consists of bacillus highland 155 and lactobacillus brevis ZG-7;
the Lactobacillus brevis ZG-7 is Lactobacillus brevis (Levilactobacillus brevis) ZG-7, which is deposited in the microorganism strain deposit center GDMCC of Guangdong in 2023, 6 and 14 days, and the deposit number is GDMCCNo.63562;
the bacillus highland 155 is bacillus highland (Bacillus altitudinis) 155 and is deposited with the microorganism strain collection center GDMCC in guangdong province at 28/2023 under the accession number GDMCC No.63563.
Further, the poultry feed additive can be optionally added with or without auxiliary materials.
Further, the auxiliary materials comprise corn flour, bean pulp and glucose.
Further, the mass ratio of the geobacillus subtilis 155 to the lactobacillus brevis ZG-7 in the composite bacteria is 1:1.
the invention also provides application of the poultry feed additive, and the poultry feed additive is used for fermenting poultry daily ration.
Further, the fermentation conditions are as follows: the temperature is 30 ℃, the humidity is 22 percent, and the fermentation time is more than 12 hours.
Further, the addition proportion of the poultry feed additive is more than 1 percent.
The invention has the following advantages: the poultry feed additive is used for fermenting poultry daily ration, the fermented daily ration can improve the growth performance of poultry, enhance the immunity of organisms, improve the antioxidation function, improve intestinal villus structure, increase the number of mucous membrane goblet cells, increase the absorption area of intestinal mucous membranes and promote the absorption of nutrient substances by small intestines.
Detailed Description
1 materials and methods
1.1 design of experiments
Healthy muscovy ducks with 800 feathers and an initial body weight (50.11+ -3.47 kg) were selected and randomly divided into 4 groups of 3 replicates each, each replicate having 10 feathers. The control group is fed with basic ration, and the feed fermentation group is added with 0% (without bacteria) and 1% and 3% of compound bacteria respectively on the basis of the basic ration, and fermented for 12 hours at room temperature (30 ℃) and 22% of humidity to prepare fermented ration. Test period 70d.
Wherein two strains in the composite bacteria are separated from the experiment (after identification and performance test of lactobacillus brevis ZG-7), and are respectively inoculated into proliferation culture media according to the inoculum size of 1 percent for respective fermentation proliferation (the culture temperature of lactobacillus brevis ZG-7 is 35 ℃, the culture temperature of bacillus stearothermophilus 155 is 33 ℃, and conventional liquid culture media are adopted for culture, such as MRS liquid culture media are adopted for lactobacillus brevis ZG-7, and the culture media adopted for bacillus stearothermophilus 155 are composed of molasses 50-10g/L, bean cake powder 10-50g/L, manganese sulfate 1-3g/L, ammonium sulfate 40-100g/L, magnesium sulfate 0.5-2g/L, sodium chloride 0.5-4g/L and pH 6.5), so that the effective viable bacteria number of the two bacteria reaches 1X 10 8 CFU/ml, then according to mass ratio 1:1, mixing and preparing the composite bacteria.
The basic ration refers to the complete compound feed prepared by the nutritional requirements of the NRC (2012) 1-70 days old muscovy ducks. The specific basal ration compositions and nutrient levels are shown in Table 1. Crude protein and dry matter measurement is carried out according to national standards GB/T6432-2018 and GB/T6438-2007; the determination of calcium and total phosphorus is referred to national standards GB/T6436-2018 and GB/T6437-2018. Diet metabolic energy (MJ/kg) = [ total feeding energy (MJ) -fecal energy (MJ) ]/diet intake (kg). Test period 75d, including a 5d pre-test period.
Table 1 basic diet composition and nutrient level (air-dried basis)
1) The premix may be provided for each kilogram of base ration: VA 12000IU; VD33600 IU; VE 20IU; vk32.4mg; VB12.0mg; vb29.0mg; 12.0mg of pantothenic acid; nicotinic acid 60.0mg; VB64.2mg; VB1230 μg; biotin 0.15mg; folic acid 1.5mg; 36.0mg of iron; copper 4.0mg; 37.5mg of zinc; 45.0mg of manganese; iodine 0.25mg; selenium 0.18mg.
2) The metabolic energy is calculated value, and the rest is measured value.
1.2 feeding management
The test muscovy ducks are fed according to a feeding mode of free feeding. 800-feather experimental ducks are fed to the same duck. Immunization, insect expelling, health care and disinfection were performed according to the conventional procedures in duck farms.
1.3 sample collection
1.3.1 blood
On the morning of the experiment, 10-feather muscovy ducks are randomly selected from each group, 5mL of blood is collected from the vein under the wing, the mixture is kept stand at room temperature for 30min, and then centrifuged for 20min at 3000r/min, and the supernatant is taken and stored at the temperature of minus 20 ℃ for later use.
1.3.2 intestinal tract and muscle tissue
At the morning of the experiment, 3 feather muscovy ducks were randomly selected for anesthesia sacrifice in each group, and the complete duodenum, jejunum, ileum, colon and cecum were collected at about 2cm, respectively. Adding 10% paraformaldehyde for fixation, dehydrating, transparentizing, waxing and embedding, and slicing by a slicer for later use. Preserving at-4 ℃ for standby.
1.4 index determination and method
1.4.1 measurement of production Performance
On days 1, 35 and 70 of the experiment, the weight was weighed on an empty stomach and recorded. Daily/group/average feed intake (ADFI) was recorded and calculated during the experiment and the experimental and control group muscovy ducks were weighed at the end of the experiment. The Average Daily Gain (ADG), feed to meat ratio (F/G) was then calculated for the four groups of muscovy ducks. The calculation formula is as follows: ADG (kg/d) = (last-first weight)/day of experiment; ADFI (kg/d) =total feed intake/experimental days; F/g=adfi/ADG.
1.4.2 blood Biochemical, immune and antioxidant index determination of Muscovy Duck
Total Protein (TP), albumin (TPT), glucose (GLU), urea Nitrogen (UN), urea (U), uric Acid (UA), triglycerides (TG), total Cholesterol (TC), high Density Lipoprotein (HDL), low Density Lipoprotein (LDL) in serum were measured using a fully automated blood biochemical analyzer (Beckmann AU5800, miami, USA). Blood immune index: immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin a (IgA) were purchased from the company of the root biotechnology (beijing) limited. Oxidation resistance index: malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) are purchased from Shanghai Biotechnology engineering services Co., ltd, and the determination method is carried out according to the operation instruction of enzyme-linked immunosorbent assay (ELISA) kit.
1.4.3 immune organ index determination
At the morning of the experiment, 3 feather muscovy ducks are randomly selected for each repetition of each group, anesthesia and sacrifice are carried out, thymus, bursa of Fabricius and spleen are respectively collected, redundant water and blood are sucked by filter paper, weighing analysis is carried out, and organ indexes are calculated:
immune organ index (mg/g) =immune organ weight (mg)/living body weight (g)
1.4.4 hematoxylin-eosin (HE) staining
Paraffin sections of each intestinal tissue prepared previously were dewaxed in sequence. And then the mixture is treated by conventional hematoxylin-eosin staining D (HE) and the like, and then the mixture is dehydrated and subjected to gum sealing. Then microscopic examination was performed, and image acquisition analysis was performed using DigiLab-C software.
1.5 statistical analysis
The test data adopts SPS19.0 to carry out single-factor analysis of variance, t test is used to carry out group comparison, the test result is represented by mean value + -standard error, P >0.05 represents that the difference is not obvious, and P <0.05 represents that the difference is obvious.
2 results
2.1 influence of the respective fermentation ration of two bacteria on the production performance of Muscovy ducks
The new single strain fermentation ration group of the feed fermentation group is divided into a lactobacillus brevis ZG-7 fermentation ration group and a bacillus altitudinis 155 fermentation ration group (1% strain is added for fermentation respectively).
As can be seen from table 2, the experiment was performed for 70 days, and the initial weight, the final weight and the daily gain of the feed fermentation group (both added and not added) were not significantly different (P > 0.05), and the average daily feed intake was significantly reduced (P < 0.05) compared to the control group. Wherein the average daily feeding of the lactobacillus brevis ZG-7 fermented ration group is 10.6g lower than that of the control group, and the difference is obvious (P < 0.05). The feed conversion ratio of the fermentation ration of the Geobacillus plateau 155 and the Lactobacillus brevis ZG-7 is also obviously reduced (P < 0.05). The feed conversion ratio of the compound bacteria fermentation ration group (1% adding proportion) is the lowest (P < 0.05), and the difference is obvious.
TABLE 2 influence of different bacteria fermented feeds on the production performance of Muscovy ducks
Note that: the same row of data in the table is marked with different lower case letters indicating significant differences (P < 0.05), and the same or no letters indicating insignificant differences (P > 0.05). The following tables are identical.
2.2 influence of two mixed bacteria fermentation ration on production performance of Muscovy ducks
As can be seen from table 3, compared with the control group, the experiment was carried out for 35 days, the initial weight, the final weight and the daily gain of the feed fermentation group (both added and not added) were not significantly different (P > 0.05), and the average daily feed intake was significantly reduced (P < 0.05). Wherein, the average daily feeding rate of the group with the addition of 3% of the two mixed bacteria fermented daily feed is 7.87g lower than that of the control group, and the difference is obvious (P < 0.05). The feed conversion also showed a significant decrease (P < 0.05). Compared with the control group, the experiment is carried out for 70 days, and the feed-meat ratio and average daily feeding of the fermented ration group are obviously reduced, wherein the addition group of 3% of two mixed bacteria is most obvious (P < 0.05). While the average daily gain and last weight differences between groups were not significant (P > 0.05).
TABLE 3 influence of two mixed bacteria fermented feeds on the production performance of Muscovy ducks
Note that: the same row of data in the table is marked with different lower case letters indicating significant differences (P < 0.05), and the same or no letters indicating insignificant differences (P > 0.05). The following tables are identical.
2.2 influence of two mixed bacteria fermentation ration on serum biochemical indexes of Muscovy ducks
As can be seen from Table 4, the fermented ration significantly increases the albumin/globulin A/G ratio (P < 0.05). Increasing the content of triglyceride TG and total cholesterol TC in serum (P < 0.05). Wherein the content of triglyceride TG and total cholesterol TC in serum of the fermentation group added with 1% and 3% of the two mixed bacteria is significantly higher than that of the control group and 0% group (P < 0.05). Other serum biochemical indexes are not obviously different (P is more than 0.05).
TABLE 4 influence of two Mixed bacteria fermentation ration on serum Biochemical index of Muscovy Duck
2.3 influence of two mixed bacteria fermentation ration on the immune index and the antioxidant capacity of the Muscovy ducks
As can be seen from table 5, the use of the daily ration by fermentation can significantly improve the indices of thymus and bursa of the immune organs of the muscovy ducks, and the difference is significant (P < 0.05). The use of two mixed bacteria for the ration fermented feed had an exponential trend to increase the spleen of the immune organs compared to the control and the non-bacteria fermented group, but the difference was not obvious (P > 0.05). The serum levels of immunoglobulin G (IgG) and immunoglobulin a (IgM) were significantly higher in the fermented ration group compared to the control (P < 0.05), with the most significant increase in the addition of the fermented group at 1% and 3% of the two mixes (P < 0.05). The content of immunoglobulin a (IgA) in the four serum groups did not differ significantly (P > 0.05). Compared with the control, the fermented daily ration is used for feeding the muscovy ducks, the overall oxidation resistance T-AOC is higher, and the difference is obvious (P is more than 0.05). Wherein, the total antioxidant capacity T-AOC, catalase CAT and superoxide dismutase SOD of the microbial fermentation liquid material group added in 3% is the highest, and the difference is obvious (P < 0.05). Four groups of malondialdehyde MDA and glutathione peroxide GSH-Px in the serum of the Muscovy ducks have no obvious difference (P is more than 0.05).
TABLE 5 influence of two Mixed bacteria fermented ration on the immune organ index of Muscovy ducks
TABLE 6 influence of fermentation of two Mixed bacteria on serum immune and antioxidant index of Muscovy Duck
2.4 influence of two mixed bacteria fermentation ration on intestinal morphology and structure of Muscovy ducks
As can be seen from Table 7, the jejunum, colon and cecum villus heights were all longer (P < 0.05) in the group with the two mixed bacteria added compared to the control group, with 3% of the two mixed bacteria added to ferment the ration group most significantly (P < 0.05). The ileal crypt height was decreased and the cecal crypt height was increased compared to the control, all with a significant difference (P < 0.05). The ratio of the heights of jejunum and colon villus to the depth of crypt of the two mixed bacteria fermented ration groups is obviously increased (P is less than 0.05), and the V/C ratio of duodenum, cecum and ileum in each group is not obviously different (P is more than 0.05).
TABLE 7 influence of fermentation of two Mixed bacteria on intestinal morphology of Muscovy Duck
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2.5 influence of two mixed bacteria fermentation ration on intestinal mucosa state of Muscovy ducks
The goblet cell is a typical mucus cell, the components of the goblet cell are mainly mucin, mucin particles are secreted outside cells, and are adhered to the surface of intestinal mucosa by synthetic mucus such as water, inorganic salt and the like to form an intestinal mucus layer, so that the goblet cell has lubricating and protecting effects and participates in the occurrence and development of various intestinal diseases. As can be seen from table 8, the fermented daily feed significantly increased the number of goblet cells on the intestinal mucosal surfaces of the ileum and colon of the muscovy duck compared to the control group, wherein the number of goblet cells on the intestinal mucosal surfaces of the ileum and colon added with the two mixed bacteria groups was significantly different (P < 0.01) compared to the control group and the non-added group. The differences were also evident in the non-added fermented groups compared to the control (P < 0.05). Compared with the control, the cup-shaped cell number of the intestinal mucosa surface of the cecum of the two mixed bacteria fermentation ration groups tends to be increased, wherein the 3% addition group has higher increasing trend, but the difference is not obvious (P is more than 0.05). The difference in the number of goblet cells at the duodenal mucosal surface was not significant (P > 0.05) between the groups.
TABLE 8 influence of two mixed bacteria fermentation ration on muscovy duck intestinal mucosa goblet cells
2.6 in vitro antibacterial action of two Mixed bacterial supernatants
The in vitro antibacterial effect of the two mixed bacteria is evaluated by using an agar diffusion test, and the result proves that the two mixed bacteria can inhibit staphylococcus aureus, shigella flexneri and salmonella typhimurium. Wherein the effect of inhibiting salmonella typhimurium is most obvious, the inhibition zone reaches 10.6+/-0.5 mm, and the difference between the inhibition zone and the inhibition zone of shigella flexneri (9.7+/-1.1 mm) is obvious compared with staphylococcus aureus (9.3+/-1.2 mm) (Table 9).
TABLE 9 antagonism of two mixed strains against 3 pathogenic bacteria of different food sources
Discussion 3
3.1 Effect of two mixed bacteria fermentation ration on Muscovy Duck production Performance
The fermented feed is used as a novel biological feed, can obviously improve the production performance of poultry, increase daily gain, reduce feed conversion ratio and increase the economic benefit of cultivation. Before, research reports that the fermented feed is fed to broiler chickens respectively, and the result shows that the fermented feed can obviously improve the feed intake and daily gain of the broiler chickens, and the feed-meat ratio is obviously reduced. In addition, research reports that the daily gain and the feed-meat ratio can be obviously increased by feeding the cherry valley ducks with the fermented feed, but the daily average feed intake has no obvious influence. The result of the invention shows that when the mixed bacteria are added into the daily ration to ferment the daily ration for 35 days, the daily average feed intake and the feed conversion ratio are obviously reduced, and the daily average weight gain is not obviously changed. When fed to 70 days of age, the daily average feed intake and feed conversion ratio continuously decrease.
In addition, the experiment shows that the daily feed intake and the feed conversion ratio of the feed fermentation group are obviously reduced along with the increase of the quantity of the two mixed bacteria during fermentation, but the daily gain is not obviously increased. This may be the case when the two mixed bacteria ferment the feed, promoting the decomposition and metabolism of the feed, and improving the gastrointestinal digestion and absorption rate of the crude protein, crude fiber and neutral washing fiber in the feed. In addition, the fermented feed has certain moisture, so that the muscovy ducks eat the feed with equal weight, but the feed with unequal amount (less feed intake) is adopted. Its growth and development are unchanged, and there is no obvious difference in daily gain. In addition, the feed conversion ratio is obviously reduced, on one hand, the feed intake of the Muscovy ducks is reduced, and on the other hand, a large amount of metabolic substances such as amino acid, vitamin, active peptide, digestive enzyme, organic acid and the like are possibly generated in the microbial fermentation process, and the active substances can promote the digestion and absorption of intestinal nutrient components, improve the conversion efficiency of the feed, and finally reduce the feed conversion ratio and increase the cultivation economic benefit.
3.2 influence of two mixed bacteria fermentation ration on serum biochemical indexes of Muscovy ducks
The biochemical index detection in the serum of the Muscovy duck can reflect the protein metabolism and fat metabolism of the Muscovy duck in real time, and simultaneously reflect the endocrine condition of the Muscovy duck and the nutrition level of daily ration. Wherein the concentration of the serum total protein can effectively reflect the anabolism strength of the protein of the organism. The invention discovers that the two mixed bacteria fermented daily ration obviously increases the concentration of the total protein of the serum of the duck, increases the concentration of the total protein of the serum, promotes the sediment of more protein in the body of the muscovy duck to tissue protein, promotes the growth and development of tissue organs of the muscovy duck, and the result shows the improvement of the production performance.
Cholesterol and triglycerides in serum are indicators of lipid metabolism in the body, and sufficient amounts of cholesterol and triglycerides are critical to the growth and development of the body. The experiment shows that the two mixed bacteria fermented ration groups obviously increase the cholesterol and triglyceride content in serum, wherein the increment of 1% and 3% additive groups is the largest. The two mixed bacteria fermented daily ration can promote the metabolism and synthesis of fat, and plays a positive role in improving the production performance.
3.3 influence of two mixed bacteria fermentation ration on the immune index and the antioxidant capacity of the Muscovy ducks
The blood immune index reflects the immune status of the Muscovy ducks. Wherein IgG plays an important role in humoral immune response to control bacterial infection in vivo, while IgA is a mucosal immune antibody of IgG only, which is the key of mucosal immunity of the organism, mainly produced by mucosal epithelium of gastrointestinal tract, and can bind various pathogenic antigens to control diarrhea infection at key time. Previous studies reported that after feeding chickens, geese and cherry valley ducks with fermented feed, after a period of time, the concentrations of IgG and immunoglobulin M (IgM) in the blood increased significantly. It has also been reported that when the fermented feed is fed to meat ducks, the serum IgG level is low, and the serum levels of immunoglobulin IgA and IgM are not different. The results of the invention show that IgG and IgM in blood of the Muscovy ducks fed with the two mixed bacteria fermented feeds are obviously increased, especially the increase of IgG is most obvious, and IgA has no obvious change. Previous studies have shown that fatty acids including monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and SFA have not only an effect of improving meat quality, but also an effect of regulating hormone metabolism and activities of various enzymes of the body, and have a wide range of effects in regulating lipid metabolism, preventing cardiovascular and cerebrovascular diseases, delaying immune function decline, and the like. Thus, the present invention shows a significant increase in IgG and IgM, which may be closely related to the large amount of fatty acids produced after microbial fermentation.
Free radicals are often used in the biological field as a surrogate for aging and disease and are subject to human beings. Thus, as an indication of the body's ability to scavenge free radicals: the total antioxidant capacity is often used as one of the health indicators of the body. GSH-Px, T-SOD and CAT are the most important enzymatic antioxidant systems of organisms, and the main functions of the GSH-Px, T-SOD and CAT are to remove excessive free radicals and active oxygen produced by the organisms and prevent lipid peroxide production. The experiment shows that when the feed is fed with the microbial fermentation feed disclosed by the invention, the T-OA content of the muscovy ducks is obviously increased, which is probably that the microorganisms generate various antioxidant active substances such as vitamins, amino acids, polyphenols and the like in the fermentation process, so that the antioxidant capacity of organisms is improved.
3.4 influence of two mixed bacteria fermentation ration on intestinal morphology and structure of Muscovy ducks
It is reported that fermented feed changes the morphology of the gastrointestinal tract compared to unfermented feed. Previous studies reported that poultry fed fermented feed had significantly longer flocks and greater flock/crypt ratios. Small intestinal villus is the fastest growing tissue of chicken and duck, and various nutrients required by the growth are directly absorbed from intestinal tracts. Previous studies found that short term "starvation" of the small intestine, with the result that the small intestine villus length was rapidly decreased, which in turn resulted in a disturbance of intestinal absorption. The microbial fermentation feed solves the problem that solid feed is often poor in palatability, causes a short-time starvation state of small intestine, can provide proper nutrients for muscovy ducks, maintains the growth of intestinal villus, and improves the feed intake.
3.5 influence of two mixed bacteria fermentation ration on intestinal goblet cells of Muscovy ducks
The cup-shaped cells of the intestinal mucosa are typical mucus cells, the components of the cup-shaped cells are mainly mucin, mucin particles are secreted outside cells, and are adhered to the surface of the intestinal mucosa through synthetic mucus such as water, inorganic salt and the like to form an intestinal mucus layer, so that the cup-shaped cells have lubricating and protecting effects and participate in the occurrence and development of various intestinal diseases. The test result shows that the number of jejunum and colon goblet cells of the fermented ration group added with the two mixed bacteria is extremely higher than that of the control group at the age of 70 days; of these, the 3% added group showed the most significant increase in intestinal mucosal goblet cells. The analysis reasons may be that two mixed bacteria ferment, a large amount of beneficial micromolecular substances such as amino acid, vitamins, active peptide, digestive enzyme, organic acid and the like are secreted, intestinal mucosa is protected, growth of intestinal villi is promoted, beneficial microorganisms in the fermented ration can be used as intestinal mucosa barriers together with mucin, pathogenic bacteria are hindered from entering the body, and the integrity of goblet cells can be regulated. In addition, some small-molecule beneficial metabolites in the fermentation product can freely pass through the mucus layer, so that the absorption of organisms is facilitated.
Conclusion 4
1) The difference in Average Daily Feed Intake (ADFI) over the whole period was insignificant in the groups with and without the two mixed bacteria fermented diets, the difference in Average Daily Gain (ADG) over the whole period and feed to meat ratio (F/G) was significant (P < 0.05), with the F/G difference being most significant in the 1% and 3% added groups (P < 0.05).
2) Compared with the control, the feed fermentation group obviously improves the total antioxidant capacity (P is less than 0.05) of serum, enhances the immunity and immune organ index, and especially has the most obvious effect in a 3% addition group.
3) Lactobacillus is added into daily ration for fermentation, so that the villus height of the colon and the cecum of the muscovy ducks is obviously improved, and the villus height/crypt depth (V/C) of the cecum is obviously improved (P is less than 0.05), wherein 3% of the daily ration is most obvious (P is less than 0.05). 4) Compared with the control group, the two mixed bacteria fermentation groups obviously increase the number of the goblet cells between the jejunum and the colonic mucosa epithelium (P < 0.05), wherein the number of the goblet cells between the jejunum and the colonic mucosa epithelium of the 3% addition group is obviously higher than that of the control group and the 0% addition group (P < 0.05).
In conclusion, the invention can obviously increase the colon and jejunum villus height, part villus/crypt ratio and goblet cell number of intestinal mucosa of the Muscovy duck through fermentation of the two mixed bacteria, improve the oxidation resistance and immunity in blood of the Muscovy duck, and improve the production performance of the Muscovy duck.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
The method comprises the following steps: identification and performance test of Lactobacillus brevis ZG-7 strain
1 isolation and determination of Lactobacillus brevis ZG-7
The strain was extracted from kimchi of tibetan left tribute herlands in 7 months 2021, and was isolated and subsequently tested in laboratory of animal husbandry and veterinary research institute of the national academy of agricultural sciences of Fujian province.
1.1 identification of Lactobacillus brevis ZG-7
(1) Morphological feature observations
Strains on MRS medium plates were picked and re-streaked onto MRS plates and incubated at 35℃for 3d to observe colony morphology.
(2) Molecular biological identification
The culture medium is removed by using PBS (phosphate buffer solution), the bacterial genome DNA of the bacterial strain is extracted by using a bacterial genome DNA extraction kit, the extracted genome DNA is used as a template, a general primer 5'-GGGGCAACCCAGCAGTTTTA-3' for fungus amplification is utilized, and a downstream primer R:5'-TAATGAGATGTTTCAGTTCACAGCG-3', the product size was 91bp gene for PCR amplification. PCR reaction system: 2×TaqPCRMix12.5. Mu.L, F primer (10. Mu. Mol/L) 1.0. Mu.L, R primer (10. Mu. Mol/L) 1.0. Mu.L, DNA template 1.5. Mu.L, ddH2O 9.5. Mu.L. PCR reaction conditions: 98 ℃ for 5min;95℃30s,55℃30s,72℃1min,35 cycles; and at 72℃for 10min. The PCR amplified product was purified and sequenced by Boshang Biotechnology Co.
1.2 antibacterial Spectrum measurement of Lactobacillus brevis ZG-7 strain supernatant
The antibacterial activity of the strain is measured by adopting an agar diffusion method. Taking supernatant of lactobacillus brevis ZG-7 fermentation broth, placing the supernatant at 37 ℃ for culture for 6 hours, observing and measuring the change of the diameter of a bacteriostasis ring by using a vernier caliper.
1.3 determination of stability of Lactobacillus brevis ZG-7 Strain supernatant against Salmonella typhimurium
(1) Acid-base stability
Taking 15mL of lactobacillus brevis ZG-7 fermentation broth supernatant, respectively regulating the pH value by using lmol/L HCl and lmol/L NaOH, reacting for 12 hours, taking salmonella typhimurium as an indicator bacterium, taking fermentation stock solution (pH3.3) as a reference, measuring the bacterial inhibition activity of the strain by adopting an agar diffusion method, repeating for 3 times, and evaluating the acid-base stability of the lactobacillus brevis ZG-7 supernatant according to the diameter change of a inhibition zone.
(2) Thermal stability
15mL of lactobacillus brevis ZG-7 fermentation broth supernatant is respectively treated for 1h at 35, 50, 65, 80, 95 and 110 ℃, salmonella typhimurium is used as an indicator bacterium, fermentation stock solution (25 ℃) is used as a control, and the antibacterial activity of the strain is measured by an agar diffusion method and is repeated for 3 times. And (5) examining the thermal stability according to the diameter change of the inhibition zone.
(3) Ultraviolet stability
15mL of lactobacillus brevis ZG-7 fermentation broth supernatant is taken, placed under a 25W ultraviolet lamp for a distance of 35cm, respectively irradiated for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours and 12 hours, salmonella typhimurium is taken as an indicator, fermentation stock solution is taken as a reference, and the antibacterial activity of the strain is measured by adopting an agar diffusion method and repeated for 3 times. And (5) inspecting the ultraviolet stability according to the diameter change of the inhibition zone.
1.4 test of Lactobacillus brevis ZG-7 strain supernatant for inhibiting Salmonella typhimurium
50 BALB/c mice of 7 days old were divided into 5 groups of blank group, lactobacillus brevis ZG-7 treated group, lactobacillus brevis BNCC treated group, lactobacillus brevis YXJ treated group and control group, each group comprising 10 male and female halves. The blank group was perfused with the supernatant of the equivalent amount of MRS medium. The control group was first drenched with 0.5mL of newly proliferated Salmonella typhimurium (concentration 10) 7 CFU/mL), and then the supernatant of the MRS medium was taken 3 times, 1mL each time, with an interval of 8h in between. Test groups were first drenched with 0.5mL of newly proliferated Salmonella typhimurium (concentration 10) 7 CFU/mL), immediately after 3 times of Lactobacillus brevis ZG-7 supernatant, lactobacillus brevis BNCC supernatant, lactobacillus brevis YXJ supernatant, 1mL each time, 8h interval. Mice diarrhea and mortality were observed and recorded.
1.5 data analysis
Data analysis was performed using Excel2017, SPSS19.01 and graphpadprim9.0.0 (121) software.
2 results
2.1 identification results of Lactobacillus brevis ZG-7 Strain
Extracting genome DNA of lactobacillus brevis ZG-7 strain, using it as template, utilizing general primer of bacteria to make amplification and sequencing of its gene sequence so as to obtain the sequence whose length is 1300bp, and submitting it to GenBank database. After BLAST comparison in NCBI database, the similarity of the DNA gene sequence of the strain and the gene sequence of the Lactobacillus brevis strain is found to be 85%, and the strain is identified as a novel Lactobacillus brevis strain by combining morphology and physiological and biochemical characteristics.
Inoculating Lactobacillus brevis ZG-7 on MRS plate, activating for 2-3 times to obtain effective viable count of 10 8 CFU/mL or more. Then inoculated into MRS medium at 2% inoculum size, shake-cultured at 35℃and 190r/min for 3d. Collecting fermentation liquor, centrifuging for 20min at 12000r/min, and filtering to obtain strain fermentation liquor supernatant.
2.2 bacteriostasis test of supernatant of Strain
As shown in Table 1, lactobacillus brevis ZG-7 can inhibit Salmonella typhimurium, and has the most obvious inhibition effect, and the inhibition zone reaches 18.9+ -1.5 a mm, compared with the common Lactobacillus brevis BNCC1853929 (BNC, purchased from North Nanoea) (5.4.+ -. 1.2 mm), lactobacillus brevis YXJ0032 (YMJ) (6.9.+ -. 1.3 mm) and Bacillus (Bacillus and Lactobacillus brevis YMJ purchased from Shanghai Yingxin laboratory), the difference of inhibition zone was obvious (3.2.+ -. 1.1) b mm)。
TABLE 1 antagonism of three different strains against Salmonella typhimurium
Note that: the different lower case letters after the data indicate significant differences at P <0.05 levels.
2.3 antibacterial stability of the supernatant of Strain
2.3.1 acid-base stability
As shown in Table 2, the supernatant of the fermentation broth of Lactobacillus brevis ZG-7, which is treated with acid and alkali at different pH values, shows a strong inhibitory activity, and the supernatant of Lactobacillus brevis ZG-7 shows a strong inhibitory activity at a low acidity, and the diameter of the inhibition zone is as high as 18.9+ -1.1, which is larger than that of the control group (supernatant when untreated), probably due to the superposition of the inhibitory effect of the acidity on Salmonella typhimurium. When the pH value is in the range of 5.3-7.3, the diameter of the inhibition zone is not obviously changed from that of the untreated group. The supernatant of the Lactobacillus brevis ZG-7 shows stronger antibacterial effect under different pH values, and the antibacterial substance has a certain acid and alkali resistance.
Acid-base stability of the supernatant of the strain of Table 2 against Salmonella typhimurium
Note that: the same row of data in the table has the same shoulder marks or no letters indicating that the difference is not significant (P > 0.05), and different lower case letters indicating that the difference is significant (P < 0.05). The following tables are identical. Ph=3.3 is the PH of the ZG-7 stock solution, here as a control.
2.3.2 thermal stability
As shown in Table 3, the supernatant of the fermentation broth of Lactobacillus brevis ZG-7 was subjected to heat treatment at different temperatures, and showed a strong antibacterial effect, which was slightly weakened with the increase in the temperature of the supernatant of Lactobacillus brevis ZG-7, wherein the diameter of the antibacterial zone of the supernatant of Lactobacillus brevis ZG-7 subjected to heat treatment at 110℃against Salmonella typhimurium was 96.03% (the reduction of 3.97%) of the control group. Therefore, the heat treatment has little influence on the bacteriostasis of the supernatant of the lactobacillus brevis ZG-7, and the bacteriostasis substance has certain thermal stability.
TABLE 3 thermal stability of Lactobacillus brevis ZG-7 supernatant against Salmonella typhimurium inhibition
2.3.3 UV stability
As can be seen from Table 4, after the supernatant of the fermentation broth of Lactobacillus brevis ZG-7 was irradiated with ultraviolet light for different periods of time, the antibacterial effect was slowly decreased with the increase of the irradiation time. The result proves that the supernatant of the lactobacillus brevis ZG-7 can relatively stably maintain the antibacterial activity under the condition of ultraviolet irradiation at a certain level.
TABLE 4 ultraviolet light stability of Lactobacillus brevis ZG-7 supernatant on Salmonella typhimurium inhibition
2.4 reduction of diarrhea and mortality in mice from strain supernatant
In order to further verify the inhibition effect of Lactobacillus brevis ZG-7 on Salmonella typhimurium, 50 mice of 7 days old were selected and divided into a blank group, a Lactobacillus brevis ZG-7 treatment group, a Lactobacillus brevis BNCC treatment group, a Lactobacillus brevis YXJ treatment group and a control group, wherein each group comprises 10 mice, each half of male and female, and the mice are infused in stomach mode.
As a result, the mice in the blank group have no diarrhea or death, and have normal feeding and drinking. Mice in the Lactobacillus brevis ZG-7 treatment group only showed slight diarrhea symptoms at the beginning of 12 hours, 1, and with continuous infusion of Lactobacillus brevis ZG-7 supernatant, mice showed no diarrhea anymore and were in normal mental state. Whereas the Lactobacillus brevis BNCC treated group and the Lactobacillus brevis YXJ treated group showed symptoms of persistent diarrhea in 2-3 mice, respectively. The control group showed severe diarrhea, which continued until the end of the experiment.
As can be seen from the results of the mice death rate, only 1 death occurred in the Lactobacillus brevis ZG-7 treated group, while 6 deaths occurred in the control group in 84h of the experimental period, and the death rate was higher. The Lactobacillus brevis BNCC treatment group and the Lactobacillus brevis YXJ treatment group respectively show 1 death (4 deaths in total in the two groups) in 12h, 48h, 60h and 90h, and the inhibition effect of the Lactobacillus brevis ZG-7 treatment group is the most obvious.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (7)
1. A poultry feed additive, characterized in that: the strain comprises a compound strain, wherein the compound strain consists of bacillus altitudinalis 155 and lactobacillus brevis ZG-7;
the Lactobacillus brevis ZG-7 is Lactobacillus brevis (Levilactobacillus brevis) ZG-7, which is deposited in the microorganism strain deposit center GDMCC of Guangdong in 2023, 6 and 14 days, and the deposit number is GDMCCNo.63562;
the bacillus highland 155 is bacillus highland (Bacillus altitudinis) 155 and is deposited with the microorganism strain collection center GDMCC in guangdong province at 28/2023 under the accession number GDMCC No.63563.
2. The avian feed additive according to claim 1 wherein: and also comprises auxiliary materials.
3. The avian feed additive according to claim 2 wherein: the auxiliary materials comprise corn flour, bean pulp and glucose.
4. The avian feed additive according to claim 1 wherein: the mass ratio of the geobacillus subtilis 155 to the lactobacillus brevis ZG-7 in the composite bacteria is 1:1.
5. use of an avian feed additive according to any one of claims 1 to 4 wherein: the poultry feed additive is used for fermenting poultry daily ration.
6. The use according to claim 5, characterized in that: the fermentation conditions are as follows: the temperature is 30 ℃, the humidity is 22 percent, and the fermentation time is more than 12 hours.
7. The use according to claim 5, characterized in that: the addition proportion of the poultry feed additive is more than 1 percent.
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