CN117562182A

CN117562182A - Preparation method and application of synbiotic enteric microcapsule for improving growth and immunity of young animals

Info

Publication number: CN117562182A
Application number: CN202311840571.1A
Authority: CN
Inventors: 崔莲花; 严昌国
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2023-12-28
Filing date: 2023-12-28
Publication date: 2024-02-20

Abstract

The invention relates to the technical field of functional feed additives for young animals at stages, in particular to a preparation method and application of a synbiotic enteric microcapsule for improving growth and immunity of young animals. The synbiotic microcapsule feed additive comprises inulin, lactobacillus acidophilus, pediococcus acidilactici and a viable bacteria freeze-drying protective agent. The synbiotic microcapsule prepared by the invention has the effects of improving the symptoms of intestinal dysbacteriosis, regulating intestinal microecology and improving the antioxidant activity of organisms, thereby improving diarrhea of young animals. The produced product is a synbiotic product, not only can exert the health care effect of the prebiotic inulin, but also can exert the probiotic effect of the probiotic composite lactobacillus. According to the invention, sodium alginate and chitosan are used as wall materials to coat the compound lactobacillus, so that the gastrointestinal tract tolerance is improved, the targeted delivery of small intestine is better realized, and the utilization efficiency in animal bodies is improved.

Description

Preparation method and application of synbiotic enteric microcapsule for improving growth and immunity of young animals

Technical Field

The invention relates to the technical field of functional feed additives for young animals in stages, in particular to a synbiotics microcapsule feed additive for improving the growth performance and immunity performance of lambs, and a preparation method and application thereof.

Background

In recent years, with the rapid development of economy in China, the living standard of people is continuously improved, and the safety and quality of animal products are paid attention to by consumers. With the implementation of national policies such as grazing prohibition, grazing withdrawal and the like, the animal husbandry mode in China is gradually changed from the traditional grazing mode into a more standardized, large-scale and intensive barn feeding and semi-barn feeding mode. The transformation of the feeding mode, the intensive and high-density feeding and the grazing condition are compared, and the risk of livestock and poultry suffering from diseases can be greatly increased. Especially, the incidence of diarrhea of young animals is increased under the influence of animal housing environment, nutrition, pathogenic bacteria and the like, and the subsequent growth and development are seriously influenced.

The average incidence rate of the lambs of different ages in months is 13.1-18.4% under the condition of the whole house, the average death rate is 69.9-78.2%, and the incidence rate is highest in the feeding period of 1 month. The traditional cultivation mode is to use a large amount of antibiotics in animal production to prevent diseases, but continuous use of a large amount of antibiotics can cause animal organisms to generate drug resistance and drug residues, thereby affecting human health.

Currently, the microecological products on the market include three types of probiotics, prebiotics and synbiotics. The following problems exist in the application process of the microecological preparation: (1) Most prebiotics are mixtures, the ingredients are undefined, the regulation mechanism is not clear enough, the functions are undefined, and the like; (2) The live bacteria of the microecological preparation are easy to die in the transportation and storage processes, so that the probiotics are poor. (3) The microecological preparation is an oral preparation, the survival rate of viable bacteria in the digestive tracts of pigs, lambs, calves and the like is very low, and the higher viable bacteria number cannot be ensured to achieve the effect of the small intestine to play a probiotic role.

The compound lactobacillus and the prebiotics are paid attention to as a widely applied microecological preparation, and the problem of poor effect exists in a single probiotic, and the probiotics of different bacteria and the prebiotics are used in a combined way to have a synergistic effect, so that the proper compound proportion of the probiotics and the matching use of the prebiotics have a vital effect on improving the intestinal health of young animals (lambs, piglets and calves), thereby improving the production performance. But the microecological preparation which is not coated is directly fed to animals, most of the viable bacteria are inactivated under the action of gastric acid and pepsin when passing through the stomach of the monogastric animal, and the number of viable bacteria is limited when reaching the intestinal tract. The rumen, the valve stomach and the reticulum of the newborn lamb are not developed yet, the abomasum is similar to the stomach of a monogastric animal, most probiotics are inactivated, and the effect of the small intestine cannot be achieved. Therefore, the microcapsule is obtained by coating the lactobacillus and the prebiotics in the polymer compound by the microcapsule coating technology, so that the probiotic effect of the lactobacillus is improved.

Therefore, aiming at the problem commonly existing in the microecological preparations on the market, inulin is used as a prebiotic, the compound lactobacillus is used as a probiotic component, and the freeze-drying protective agent is added to prepare the enteric-coated synbiotics microcapsule, so that on one hand, the in-vivo probiotics effect of the inulin is promoted through the synergistic effect of the inulin and the compound probiotic; on the other hand, the coating technology targeting small intestine targeted delivery is utilized to carry out coating treatment on the synbiotics component so as to achieve the synbiotics and probiotics effects.

Disclosure of Invention

The technical scheme of the invention is as follows:

the invention aims to provide a preparation method and application of a synbiotic enteric microcapsule for improving the growth and immunity of young animals, in particular to a preparation method and application effect of a synbiotic microcapsule feed additive for improving the growth and immunity of lambs.

The synbiotic microcapsule feed additive consists of the following components: inulin, lactobacillus acidophilus, pediococcus acidilactici and viable bacteria freeze-drying protective agent.

The preparation method of the synbiotic microcapsule comprises the following steps:

1. preparation of wall material solution: 2-4wt% sodium alginate solution, 0.8wt% chitosan solution and 0.1wt% sodium alginate solution are prepared.

2. Preparing bacterial mud of composite lactic acid bacteria: culturing Pediococcus acidilactici and Lactobacillus acidophilus at ratio of 1:2-2:1 in MRS liquid culture medium for 12-16 hr, centrifuging for 2-3 times, washing with 0.85% physiological saline for 2-3 times, and collecting. Further, 0.85% sodium chloride solution was addedThe final viable count concentration reaches 2.5-4.3X 10 ¹¹ cfu/mL. 5mg-10mg of inulin is added into 1mL of the compound lactobacillus system, and the mixture is uniformly mixed for standby.

3. The freeze-drying protective agent uses glycerol and corn starch, and the concentration of the freeze-drying protective agent reaches 2-10%.

4. The concentration of calcium chloride is formulated to be 0.2-0.5M.

5. Mixing the bacterial sludge in the step (2) with the sodium alginate solution prepared in the step (1) by utilizing an extrusion method to ensure that the probiotics content reaches 20-40%, further dripping the mixture into 0.2-0.5M calcium chloride solution to perform gelation reaction, standing for 5-30min at the temperature of 20-30 ℃ to form calcium alginate microspheres, and forming a first layer of coating;

washing the calcium alginate microspheres prepared in the step (5) with 6.0.85% physiological saline;

7. pouring the microspheres prepared in the step (6) into chitosan solution, reacting for 5-30min, and flushing with 0.85% physiological saline for 2-3 times to form a second layer of coating.

8. Pouring the microspheres prepared in the step (7) into sodium alginate solution, reacting for 5-30min, and washing with 0.85% physiological saline for 2-3 times to form a third layer of coating. Finally, the freeze dryer freeze-dries the microcapsules.

The synbiotic microcapsule prepared by the invention is subjected to three-layer coating treatment, and has the effects of improving symptoms of intestinal flora imbalance, regulating intestinal microecology, improving the antioxidant activity of organisms, and further improving diarrhea of young animals. The produced product is a synbiotic product, not only can exert the health care effect of the prebiotic inulin, but also can exert the probiotic effect of the probiotic composite lactobacillus. The sodium alginate and chitosan are used as wall materials to coat the compound lactobacillus, so that the gastrointestinal tract tolerance is improved, the targeted delivery of small intestine is better realized, and the utilization efficiency in animal bodies is improved.

The growth performance of the lambs is obviously improved by the invention 1); 2) In terms of apparent digestibility of the feed, compared with a control group, the digestibility of crude protein in the T4 group is improved by 11.48 percent (p < 0.05); the apparent digestibility of calcium in the T4 group is improved by 8.95 percent compared with the NC group, the apparent digestibility of phosphorus is improved by 10.29 percent (p < 0.05), and the neutral washing fiber digestibility of the T4 group is improved by 12.17 percent (p < 0.05) compared with the CON group; 3) From lamb blood cell analysis, the synbiotic microcapsule fed with lactobacillus and inulin has the main effects that the average hemoglobin content of the T4 group is obviously improved (p is less than 0.05) compared with the control group in the aspect of blood biochemical indexes, and the hemoglobin in the organism has the main effects of carrying oxygen through blood circulation, so that the oxygen can be transported to various parts of the organism to perform normal vital activities, and the metabolism of animals is improved; 4) From the analysis of the antioxidant and anti-stress indexes of the lamb serum, compared with other groups, the blood of the T4 group of lambs has obvious SOD content (p is less than 0.05), the T-AOC, GSH-PX and NOS content of the T4 group and the T3 group of lambs are obviously improved (p is less than 0.05), the MDA content of the T4 group of lambs is obviously reduced (p is less than 0.05), and the MPO content of the T3 group and the T4 group of lambs is obviously reduced (p is less than 0.05). The synbiotics of the invention can effectively improve the anti-oxidative stress capability of the lamb animals, and is helpful for improving the anti-stress capability of the animals. 5) And in the aspect of lamb serum inflammatory factor index, compared with a control group, the indexes of IL-6, TNF alpha and IL-4 of a T3 group and a T4 group fed with the synbiotic microcapsule of lactobacillus and inulin are obviously improved (p < 0.05). 6) Analysis of immunoglobulin in lamb blood, the T3 and T4 groups showed significantly higher immunoglobulin content (p < 0.05) in the blood compared to the control and empty capsule groups. According to the invention, the sodium alginate/chitosan microcapsule which has smaller particle size, uniform distribution, regular spherical shape, smooth surface, larger production amount and better activity of the embedded material is prepared by improving the microencapsulation method.

Drawings

FIG. 1 shows a comparison of the freeze-drying protection rate of Pediococcus acidilactici microcapsules;

FIG. 2 shows SEM observations of Pediococcus acidilactici microcapsule morphology features;

FIG. 3 shows the in vitro analysis of the tolerance of plant starch to Pediococcus acidilactici in artificial gastrointestinal fluids, FIG. 3 (A) SGF (without pepsin); FIG. 3 (B) SGF containing pepsin); FIG. 3 (C) SGF (without bile salts); FIG. 3 (D) SGF (bile salts)

Detailed Description

The following examples are given to illustrate the present invention and are not to be construed as limiting its scope, since they are intended to cover any unnecessary modifications and variations of the present invention that would occur to those skilled in the art in light of the above teachings.

The synbiotic microcapsule feed additive disclosed by the invention consists of the following components: inulin, lactobacillus acidophilus, pediococcus acidilactici and viable bacteria freeze-drying protective agent.

2. Preparing bacterial mud of composite lactic acid bacteria: culturing Pediococcus acidilactici and Lactobacillus acidophilus at ratio of 1:2-2:1 in MRS liquid culture medium for 12-16 hr, centrifuging for 2-3 times, washing with 0.85% physiological saline for 2-3 times, and collecting. Further, 0.85% sodium chloride solution is added to make the final viable count concentration reach 2.5-4.3X10 ¹¹ cfu/mL. 5mg-10mg of inulin is added into 1mL of the compound lactobacillus system, and the mixture is uniformly mixed for standby.

4. The concentration of calcium chloride is formulated to be 0.2-0.5M.

Pediococcus acidilactici (Pediococcus acidilactici, PA) of the invention are all wild type and provided by the animal nutrition laboratory of the university of Yanbian, agricultural college. MRS liquid medium, MRS agar medium, PBS, available from Beijing Soy Bao technology Co., ltd; potato starch, corn starch, chitosan, calcium chloride dihydrate were purchased from microphone agents; all others are domestic analytically pure reagents.

Example 1

Preparing a wall material solution by preparing a sodium alginate solution with the concentration of 2wt%, a chitosan solution with the concentration of 0.8wt% and a sodium alginate solution with the concentration of 0.1 wt%. Pediococcus acidilactici and Lactobacillus acidophilus are cultured in MRS liquid culture medium for 12 hr, centrifugated for 3 times, washed with 0.85% physiological saline for 3 times, and collected. Further, 0.85% sodium chloride solution was added to achieve a final viable cell count concentration of 4.3X 10 ¹¹ 5mg-10mg of inulin is added into the cfu/mL and 1mL of compound lactobacillus system, and the mixture is uniformly mixed for standby. Glycerin and zein were used as lyoprotectants to achieve a concentration of 2%. Further, the calcium chloride concentration was formulated to be 0.2M. Mixing the bacterial mud with the sodium alginate solution by using an extrusion method to ensure that the probiotics content reaches 40%, further dripping the mixture into 0.2M calcium chloride solution by using a syringe to perform gelation reaction, wherein the temperature is 20 ℃, and the standing time is 30min to form calcium alginate microspheres; washing the calcium alginate microspheres by using 0.85% physiological saline to form a first layer of coating; pouring the prepared microsphere into chitosan solution, reacting for 30min, and washing with 0.85% physiological saline for 3 times to form a second layer of coating. Pouring the prepared microsphere into sodium alginate solution, reacting for 30min,0.85The third coating was formed by washing 3 times with% physiological saline. Finally, the freeze dryer freeze-dries the microcapsules.

Example 2

The test groups were selected from sodium alginate 3.0%, chitosan 0.8%, potato starch 10%, hardening time 10min (T1 group) and sodium alginate 3.0%, chitosan 0.8%, corn starch 10% (T2 group). The sodium alginate concentration is 3.0%, the chitosan concentration is 0.8%, and the hardening time is 10min, which is the control group (CON group). Firstly, the cultured wild type PA is centrifuged at 2000r/min for 10min at 4 ℃, the particles are washed, and the activated PA bacterial sludge (7.53×10) ¹¹ CFU/mL) was suspended in 10mL of physiological saline to prepare a bacterial solution. Then mixed with 3% sodium alginate and 2% glycerol. And respectively adding potato starch and corn starch. Dripping 0.2M CaCl at uniform speed by a disposable sterile syringe ₂ And (3) magnetically stirring for 10min to completely harden the microcapsule, and then washing with 0.85% physiological saline to obtain the single-layer microcapsule. The single-layer microcapsule is placed in 0.8% chitosan solution and stirred for 10min to form a film, so as to prepare the sodium alginate/chitosan double-layer microcapsule, and finally the double-layer microcapsule is completely washed by 0.85% normal saline, stored for 24h in a refrigerator at the temperature of minus 80 ℃ and then is placed in a vacuum freeze dryer for freeze drying.

Experimental example

The synbiotic microcapsules prepared in example 1 were used in lamb production and were specifically operated as follows:

1. animal implementation: 25 hybrid lambs of Wu Zhumu which are 10 days old, consistent in genetic background, good in health condition and close in weight (5.39+/-0.29) kg after birth are selected and randomly divided into 5 groups of 5 lambs.

2. And (3) test design: the test components were 5 groups comprising:

namely, a control group (CON group) is fed with basic ration, and an empty capsule group (T1 group) is fed with basic ration plus 16mg of empty capsules; the uncoated group (T2 group) was fed with 100 mg/dose of base ration, uncoated lactic acid bacteria and inulin (viable count: 2.0X10) ⁸ CFU/g), microcapsule low dose group (T3 group) fed basal diet + microcapsules coated with lactobacillus combined inulin 20 mg/only (viable count: 2.0X10 ⁸ CFU/g); basic ration + milk for feeding of microcapsule high-dose (T4 group) group200 mg/dose (viable count: 2.0X10) of acid bacteria combined inulin microcapsule ⁹ CFU/g)。

In the present invention, the measurement index includes:

(1) Average daily gain: each lamb was weighed on an empty stomach before feeding on day 1, day 30 and day 60 morning and the weight gain on a 60 balance was calculated.

(2) Average daily feed intake of the lambs was calculated: the feed intake of the lambs was calculated for 7 consecutive days of the test period and repeated 2 times, thereby calculating the average daily feed intake.

(3) Material weight ratio: calculated according to the average daily feed intake and the average daily weight gain.

(4) Apparent digestibility:

apparent digestibility of nutrient = [ (nutrient amount fed-nutrient amount in manure)/nutrient amount fed ] ×100%

(5) Sample collection: the fasting carotid artery blood sampling of the experimental sheep before feeding on the 60 th morning is analyzed for serum biochemical index, antioxidant index, immune index and the like.

(6) Data statistics: test data were collated with Excel2007, single-factor analysis of variance with SPSS 27.0, multiple comparisons with Duncan's method, p <0.05 were significant differences.

The effect of the lactobacillus-inulin-combined synbiotic microcapsules on the growth performance of the lambs is shown in table 1, and the effect of the lactobacillus-inulin-combined synbiotic microcapsules on the apparent digestibility of the lamb feed is shown in table 2. The effect of the lactobacillus in combination with inulin synbiotic microcapsules on lamb blood cells is shown in table 3. The effect of the lactobacillus in combination with inulin on the biochemical index of lamb blood is shown in Table 4. The effect of the lactobacillus in combination with inulin on the index of antioxidant stress in lamb blood is shown in Table 5. The effect of the lactobacillus in combination with inulin synbiotic microcapsules on the blood inflammation index of lambs is shown in table 6. The effect of the lactobacillus in combination with inulin on the blood immune index of lambs is shown in Table 7.

(1) Lamb growth Performance analysis

As can be seen from Table 1, the weight of each group of lambs gradually increased with the increase of the age of the day; the average daily gain of the T2, T3 and T4 groups was highest in the T4 group (p < 0.05); no significant difference (p > 0.05) during the average daily feed intake test; the ratio of the material to the weight of the T3 group and the T4 group is obviously improved (p is less than 0.05).

TABLE 1 Effect of Lactobacillus-inulin-associated synbiotic microcapsules on lamb growth performance

Note that: the different letters of the same row of data shoulder marks represent significant differences (p < 0.05); the same letter or no letter indicates that the difference is not significant (p > 0.05); the table below is the same.

(2) Apparent digestibility analysis of lamb feed

Feeding lactobacillus and inulin synbiotic microcapsule improves the apparent digestibility of the lamb feed by 11.48 percent (p < 0.05) compared with the control group; compared with NC group, the apparent digestibility of T4 group calcium is improved by 8.95%, the apparent digestibility of phosphorus is improved by 10.29% (p < 0.05); the neutral wash fiber digestibility of the T4 group was increased by 12.17% (p < 0.05) compared to the CON group.

TABLE 2 Effect of Synbiotic microcapsules of lactic acid bacteria in combination with inulin on apparent digestibility of lambs

(3) Lamb blood cell analysis

Compared with a control group, the synbiotic microcapsule fed with lactobacillus and inulin has the advantages that the average hemoglobin content of the T4 group is obviously improved (p is less than 0.05) in terms of blood biochemical indexes, and the main effect of hemoglobin in the organism is that oxygen is carried through blood circulation, so that the oxygen can be carried to various parts of the organism to perform normal vital activities, and the metabolism of animals is improved.

TABLE 3 influence of Synbiotic microcapsules of lactic acid bacteria in combination with inulin on the biochemical index of lamb blood

(4) Analysis of physiological and biochemical indexes of lamb serum

The synbiotic microcapsule fed with lactobacillus and inulin has all indexes in normal range in the aspect of blood physiological and biochemical indexes of lambs, and no obvious difference (p > 0.05) among groups.

TABLE 4 influence of Synbiotic microcapsules of lactic acid bacteria in combination with inulin on the physiological and biochemical index of lamb blood

(5) Lamb serum antioxidant and anti-stress index analysis

As can be seen from Table 5, the blood SOD content of the T4 group lambs was significant (p < 0.05) compared with the other groups; compared with other groups, the T-AOC, GSH-PX and NOS contents of the T4 group and the T3 group are obviously improved (p is less than 0.05); MDA content of the T4 group was significantly reduced (p < 0.05) compared to the other groups; the MPO content was significantly reduced (p < 0.05) in the T3 and T4 groups compared to the other groups. MPO is a hemase secreted by activated neutrophils and monocyte macrophages, plays a bactericidal role in the innate immunity of the host, and is involved in the occurrence and development of inflammation. Malondialdehyde (MDA) is an oxidation product of Reactive Oxygen Species (ROS) after acting on polyunsaturated fatty acids, and is an indicator of lipid peroxidation. The synbiotics of the invention can effectively improve the anti-oxidative stress capability of the lamb animals, and is helpful for improving the anti-stress capability of the animals.

TABLE 5 influence of Synbiotic microcapsules of lactic acid bacteria in combination with inulin on the physiological and biochemical index of lamb blood

(6) Lamb serum inflammatory factor analysis

In terms of lamb serum inflammatory factor indexes, compared with a control group, the indexes of IL-6, TNF alpha and IL-4 of a T3 group and a T4 group of synbiotic microcapsules fed with lactobacillus and inulin are obviously improved (p < 0.05).

TABLE 6 Effect of Synbiotic microcapsules of lactic acid bacteria in combination with inulin on blood inflammatory factors of lambs

(7) Analysis of immunoglobulin in lamb blood

The T3 and T4 groups had significantly increased blood immunoglobulin levels (p < 0.05) compared to the control group and the empty capsule group.

TABLE 7 Effect of Lactobacillus in combination with inulin Synbiotic microcapsules on immunoglobulins in lamb blood

The growth performance of the lambs is obviously improved by the invention 1); 2) In terms of apparent digestibility of the feed, compared with a control group, the digestibility of crude protein in the T4 group is improved by 11.48 percent (p < 0.05); the apparent digestibility of calcium in the T4 group is improved by 8.95 percent compared with the NC group, the apparent digestibility of phosphorus is improved by 10.29 percent (p < 0.05), and the neutral washing fiber digestibility of the T4 group is improved by 12.17 percent (p < 0.05) compared with the CON group; 3) From lamb blood cell analysis, the synbiotic microcapsule fed with lactobacillus and inulin has the main effects that the average hemoglobin content of the T4 group is obviously improved (p is less than 0.05) compared with the control group in the aspect of blood biochemical indexes, and the hemoglobin in the organism has the main effects of carrying oxygen through blood circulation, so that the oxygen can be transported to various parts of the organism to perform normal vital activities, and the metabolism of animals is improved; 4) From the analysis of the antioxidant and anti-stress indexes of the lamb serum, compared with other groups, the blood of the T4 group of lambs has obvious SOD content (p is less than 0.05), the T-AOC, GSH-PX and NOS content of the T4 group and the T3 group of lambs are obviously improved (p is less than 0.05), the MDA content of the T4 group of lambs is obviously reduced (p is less than 0.05), and the MPO content of the T3 group and the T4 group of lambs is obviously reduced (p is less than 0.05). The synbiotics of the invention can effectively improve the anti-oxidative stress capability of the lamb animals, and is helpful for improving the anti-stress capability of the animals. 5) And in the aspect of lamb serum inflammatory factor index, compared with a control group, the indexes of IL-6, TNF alpha and IL-4 of a T3 group and a T4 group fed with the synbiotic microcapsule of lactobacillus and inulin are obviously improved (p < 0.05). 6) Analysis of immunoglobulin in lamb blood, the T3 and T4 groups showed significantly higher immunoglobulin content (p < 0.05) in the blood compared to the control and empty capsule groups.

As can be seen from fig. 1, the freeze-drying protection rate of both the T1 group and the T2 group was significantly higher than that of the control group (p < 0.05); the difference in lyoprotection rates was not significant (p > 0.05) between the T1 and T2 groups.

In the invention, two groups of microcapsules are prepared by analyzing morphological characteristics and drug loading capacity of the Pediococcus acidilactici microcapsules by using plant starch, and two groups of microcapsules are prepared by adding potato starch and corn starch, and the microcapsules without starch are used as a control group, and the morphological characteristics are analyzed, and the results are shown in Table 8. As can be seen from table 8, the microcapsule particle sizes of the T1 and T2 groups were significantly higher than the control group (p < 0.05), and the microcapsule particle size of the T1 group was the largest (p < 0.05); the microcapsules of group T2 have the greatest mechanical strength (p < 0.05); the amount of the microcapsule bacteria carried by the T1 group and the T2 group is higher than that of the control group, and the difference is obvious (p < 0.05).

TABLE 8 characterization of PA microcapsules (after lyophilization)

According to the invention, in the example 2, as shown in fig. 2, sodium alginate/chitosan microcapsules with smaller particle size, uniform distribution, regular spherical shape, smooth surface, larger production amount and better activity of the embedded substances are prepared by improving the microencapsulation method. The potato starch and the glycerol are used as the freeze-drying protective agent, and the corn starch and the glycerol are used as the freeze-drying protective agent, so that the protective effect, the mechanical strength of the microcapsule, the morphological plumpness and the tolerance in vitro gastrointestinal fluid can be improved, and the effect is obvious compared with that of a control group. However, the microcapsules of corn starch and glycerin as lyoprotectants were more plump and regular in shape, and also higher in mechanical strength, as observed from SEM. Comprehensively considering, the corn starch and the glycerol are considered to be the freeze-drying protective agent, which is more beneficial to the shape maintenance and the freeze-drying protective effect of the lactobacillus microcapsule.

As can be seen from fig. 3A, the survival rates of the pediococcus acidilactici of the CON group, the T1 group and the T2 group under the SGF condition of pH 2.0 are 60.40%, 66.19% and 64.66%, respectively, and there is no significant difference between the three groups (p > 0.05). Compared with a control group, the survival rate of the starch-added microcapsule pediococcus acidilactici is improved; as can be seen from fig. 3B, the protection rate of both T1 and T2 groups in SGF containing pepsin was higher than that of the control group, and the difference in survival rate of pediococcus acidilactici was significant (p < 0.05) for 2 h; as shown in fig. 3C, 3 groups of pediococcus acidilactici microcapsules begin to release rapidly after 10min in the artificial intestinal juice with the pH of 7.2, and at 1h, the pediococcus acidilactici microcapsules of the CON group, the T1 group and the T2 group disintegrate basically and completely, and at 4h, the release rates of pediococcus acidilactici microcapsules of the CON group, the T1 group and the T2 group reach 87.74%, 85.14% and 87.84%, respectively, the release rates of the pediococcus acidilactici microcapsules are not obvious (p > 0.05), which indicates that starch is added as a protective agent of the pediococcus acidilactici microcapsules, and the release of pediococcus acidilactici in the intestinal juice is not influenced; as can be seen from fig. 3D, the survival rate of 3 groups of microcapsules under the action of bile salt is reduced, and the survival rate of T1 and T2 groups of microcapsules of pediococcus acidilactici is significantly higher than that of the control group (p < 0.05) at 4 hours, which indicates that the addition of starch as a lyoprotectant of the pediococcus acidilactici microcapsules can enhance the resistance of pediococcus acidilactici to bile salt.

The experimental results show that the microencapsulated synbiotic microcapsule has remarkable effect on improving the growth performance and immunity of the lambs. The invention does not contain any antibiotics or other chemical medicines, and has strong practical application value in the production of young animals as an antibiotic-free product.

Claims

1. A feed additive of synbiotic microcapsule comprises inulin, lactobacillus acidophilus, pediococcus acidilactici, and viable bacteria freeze-drying protective agent.

2. The synbiotic microcapsule feed additive according to claim 1, wherein the ratio of pediococcus acidilactici to lactobacillus acidophilus is 1:2-2:1.

3. A method of preparing the synbiotic microcapsule feed additive of claim 1 comprising the steps of:

the preparation method of the wall material solution comprises the following steps: preparing 2-4wt% sodium alginate solution, 0.8wt% chitosan solution and 0.1wt% sodium alginate solution;

the preparation method of the bacterial mud of the composite lactic acid bacteria comprises the following steps: culturing Pediococcus acidilactici and Lactobacillus acidophilus at ratio of 1:2-2:1 in MRS liquid culture medium for 12-16 hr, centrifuging for 2-3 times, washing with 0.85% physiological saline for 2-3 times, collecting, and adding 0.85% sodium chloride solution to obtain final viable count concentration of 2.5-4.3X 10 ¹¹ cfu/mL, then adding 5mg-10mg of inulin into 1mL of compound lactobacillus system, and uniformly mixing for later use;

and (3) a gelation reaction step: mixing the bacterial mud with the sodium alginate solution by using an extrusion method to ensure that the probiotics content reaches 20-40%, further dripping the mixture into 0.2-0.5M calcium chloride solution to perform gelation reaction, wherein the temperature is 20-30 ℃, and the standing time is 5-30min to form calcium alginate microspheres to form a first layer of coating; washing calcium alginate microspheres with 0.85% physiological saline, pouring the microspheres into chitosan solution, reacting for 5-30min, and washing with 0.85% physiological saline for 2-3 times to form a second coating; pouring the microspheres into sodium alginate solution, reacting for 5-30min, and washing with 0.85% physiological saline for 2-3 times to form a third layer of coating;

and (3) freeze-drying: freeze-drying the microcapsule by a freeze dryer.

4. The application of the synbiotic microcapsule feed additive is used for improving the growth and immunity of young animals.