A method for feeding broiler chickens with clostridium butyricum

Abstract

The present application belongs to the field of broiler feed additives, and provides a broiler Clostridium butyricum activation feeding method. The present application reasonably utilizes the mechanical stirring anaerobic fermentation system technology customized for anaerobic fermentation, can utilize inert gas to protect the bacteria, and can activate a large number of bacteria in vitro to adapt to production; after the bacteria are activated in vitro, the bacteria can quickly play a role in the body, can reduce the loss of most probiotics caused by insufficient in-vivo activation and intestinal colonization resistance, and can improve the use effect of metabolic products, so that the feed-to-weight ratio is reduced in the actual application of broiler breeding, and therefore the present application provides a new method for the reasonable use of probiotics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of broiler feed additives, and relates to an in-vitro Clostridium butyricum microbial agent activation and feeding method for improving broiler intestinal health and increasing broiler production performance. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is publicly known.

[0003] Antibiotic residues and bacterial drug resistance increase, which brings great hidden dangers to human health. In many countries, the use of antibiotics in breeding is completely prohibited. Under this background, high-density breeding still brings various problems to broiler breeding, leading to the occurrence of some common diseases, increasing the mortality rate of chicken flocks, and ultimately causing great economic losses, which is not conducive to the development of the breeding industry. Therefore, finding green antibiotic substitutes has become an urgent need for efficient production of broiler breeding. Probiotic preparations have become an excellent choice due to their ability to improve the production performance of broilers, inhibit the growth of harmful bacteria, and adjust the balance of intestinal microecology.

[0004] Clostridium butyricum preparation has become a new research direction in the field of probiotics due to its special nutritional therapy effect on the gastrointestinal tract and strong stability. It has been confirmed that Clostridium butyricum added in feed can enter the body as a non-specific immune factor to promote the immune ability of the animal body, and produce short-chain fatty acids of beneficial intestinal flora, reduce the number of harmful bacteria, and optimize the structure of intestinal flora. Butyric acid, as one of the main metabolic products of Clostridium butyricum, can provide nutrition to maintain the function of intestinal epithelial cells, play a key role in the repair of epithelial cells, and play an important role in regulating immune and inflammatory responses and intestinal barrier function. In addition, Clostridium butyricum can secrete some enzymes such as amylase, protease, and phospholipid synthase during its growth and reproduction in the intestinal tract of livestock and poultry, which can degrade the nutrients in feed into substances such as glucose that are easier for the animal body to absorb, thereby improving the feed conversion efficiency. Some oligosaccharides produced at the same time also provide nutrients for other probiotics, promote the growth of dominant flora, and adjust the balance of intestinal microbial flora.

[0005] However, the use of probiotics currently faces the serious problem of colonization resistance. Probiotics enter the stomach through the esophagus after being eaten and are affected by the acidic gastric juice environment and mechanical agitation, but more than 90% of the strains can still pass through the pylorus into the intestine, where the environment is more suitable for the survival and colonization of probiotics. However, the bacterial density in the intestine is the highest, and probiotics must compete with the host microbiota for nutrients and adhesion sites, so most probiotics are excreted from the colon shortly after oral administration and cannot exert their effects, and therefore colonization resistance is one of the important reasons for inhibiting the long-term effects of probiotics.

[0006] Clostridium butyricum is a strict anaerobe, and as a feed additive it needs to be activated in the body before it can colonize the intestine. The short intestine of broilers and the short residence time of feed in the intestine further restrict the colonization of most Clostridium butyricum in the intestine and the production of butyric acid after entering the body, affecting the use effect. SUMMARY

[0007] To solve the above problems, the present application provides a Clostridium butyricum in vitro activation feeding method to improve the production performance of broilers and improve the intestinal health of broilers. The first objective is to improve the barrier function and intestinal flora structure of broilers, and to achieve the improvement of the intestinal function of broilers. The second objective is to provide a probiotic in vitro activation application method to reduce the loss of probiotics during colonization in the intestine, ultimately improve the production performance of broilers and improve the intestinal health, and improve the economic benefits of breeding.

[0008] To achieve the above-mentioned objectives, the present application adopts the following technical solutions:

[0009] In a first aspect of the present application, a Clostridium butyricum activation feeding method for broilers is provided, comprising:

[0010] The Clostridium butyricum is activated, inoculated into a liquid culture medium for culture and amplification, and centrifuged to obtain a bacterial strain;

[0011] The bacterial strain is pre-frozen at -20 to -18℃ for 4 to 5 hours, then placed in a -80 to -70℃ environment overnight, and then the frozen bacterial strain is placed in a pre-cooling environment at -55 to -57℃, pre-heated for 20 to 25 minutes, main drying for 15 to 18 hours, and final drying for 1 to 1.5 hours to obtain a bacterial powder;

[0012] The bacterial powder is subjected to anaerobic liquid fermentation to obtain a Clostridium butyricum fermentation broth;

[0013] The wet mixing method is used to add 10% v / w of the Clostridium butyricum fermentation broth to the normal daily ration, and then the mixture is fed.

[0014] At present, the research of Clostridium butyricum is mostly in medical application, and there is no clear research on the method of adding Clostridium butyricum in broiler feed, especially the method of combining in-vitro activation technology with wet mixing feed, mainly due to the limitation of anaerobic activation conditions, which is difficult to activate in large quantities and adapt to breeding production. The in-vitro activation technology is to pre-culture Clostridium butyricum in a liquid fermentation tank in-vitro, and then mix with feed to feed broilers. The advantages of this method are short activation period, high yield, low cost, high viable count and high activity of activated bacteria, and contains a large amount of bacterial metabolites. The present application reasonably utilizes the mechanical stirring anaerobic fermentation system technology for anaerobic fermentation, which can protect the bacteria by inert gas and activate a large amount of bacteria in-vitro to adapt to production; the bacteria activated in-vitro can quickly play a role in the body, reduce the loss of most probiotics caused by insufficient in-vivo activation and intestinal colonization resistance, and improve the use effect of metabolites, so as to reduce the feed-to-gain ratio in the practical application of broiler breeding. Therefore, the present application provides a new method for the rational use of probiotics.

[0015] In a second aspect of the present application, the in-vitro activated bacterial agent prepared by the above method is provided.

[0016] The Clostridium butyricum fermentation broth is added to normal daily ration in a certain proportion for feeding, in order to improve the production performance of broilers, and the specific operation is as follows:

[0017] 1. According to the feed formula design of broiler breeding standard, normal daily ration is prepared, and 10% Clostridium butyricum fermentation broth (v:w) is added to the normal daily ration by wet mixing method, and the mixture is stirred uniformly, and the final bacterial content in the feed is 1x10 9 CFU / kg feed.

[0018] 2. Daily feed intake is counted and re-mixed and replaced to ensure the viability of the bacterial population in the feed.

[0019] In a third aspect of the present application, the application of the above in-vitro activated bacterial agent in the preparation of products with any one of the functions of 1)-4) is provided:

[0020] 1) improving the production performance of broilers, which is reflected in any one of a1)-a2): a1) increasing average daily gain, and a2) reducing feed-to-gain ratio;

[0021] 2) improving the intestinal development of broilers, which includes: increasing the villus height of jejunum and ileum;

[0022] 3) improving the intestinal barrier function of broilers, which includes: significantly up-regulating the expression of MUC2 mRNA, Occludin mRNA or MUC5AC mRNA in jejunum;

[0023] 4) Optimizing the intestinal flora structure of broilers, including: increasing the number of OTUs unique to the flora species, increasing the flora abundance of Bacteroidetes, Proteobacteria or Faecalibacterium prausnitzii, and changing its characteristic flora.

[0024] Advantages of the present application

[0025] (1) The present application activates Clostridium butyricum in vitro by means of liquid anaerobic fermentation, and cultures to a specified concentration. On the basis of feeding a basal diet, the in-vitro-activated Clostridium butyricum is added to the diet by wet mixing, which can improve the intestinal barrier function of broilers, improve intestinal health, improve feed conversion efficiency of broilers, improve production performance of broilers, and thus increase the economic benefits of broiler farming.

[0026] (2) Compared with traditional Clostridium butyricum agents, the in-vitro-activated Clostridium butyricum agent of the present application can more comprehensively and more optimally improve the effects of broiler farming from multiple aspects such as production performance, intestinal development, intestinal barrier function and flora structure, significantly improving the intestinal barrier function, intestinal health, feed conversion efficiency and production performance of broilers.

[0027] (3) The preparation method of the present application is simple, practical and easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0028] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with examples of the exemplary embodiments of the present application and their description, explain the present application, and do not constitute an improper limitation of the present application.

[0029] Figure 1 Morphological structure of duodenum after feeding Clostridium butyricum (A: 21d, CON, 40x; B: 21d, FW1, 40x; C: 42d, CON, 40x; D: 42d, FW1, 40x);

[0030] Figure 2 Morphological structure of jejunum after feeding Clostridium butyricum (A: 21d, CON, 40x; B: 21d, FW1, 40x; C: 42d, CON, 40x; D: 42d, FW1, 40x);

[0031] Figure 3 Morphological structure of ileum after feeding Clostridium butyricum (A: 21d, CON, 40x; B: 21d, FW1, 40x; C: 42d, CON, 40x; D: 42d, FW1, 40x);

[0032] Figure 4For the effect of Clostridium butyricum on intestinal permeability of broilers (A: 21d; B: 42d), n = 10, note: a. b. Different letters in each row represent significant difference (P < 0.05).

[0033] Figure 5 For the effect of Clostridium butyricum feeding on the mRNA expression of intestinal barrier related genes of 21-day-old broilers (A: duodenum; B: jejunum; C: ileum), n = 10, note: * indicates significant difference compared with other groups (P < 0.05).

[0034] Figure 6 For the effect of Clostridium butyricum feeding on the mRNA expression of intestinal barrier related genes of 42-day-old broilers (A: duodenum; B: jejunum; C: ileum), n = 10, note: * indicates significant difference compared with other groups (P < 0.05).

[0035] Figure 7 For the Venn diagram (A: 21 days old; B: 42 days old).

[0036] Figure 8 For the heat map at the level of door classification.

[0037] Figure 9 For the heat map at the level of genus classification.

[0038] Figure 10 For the analysis of Alpha diversity index of 21-day-old broilers.

[0039] Figure 11 For the analysis of Alpha diversity index of 42-day-old broilers.

[0040] Figure 12 For the Beta diversity PCoA analysis.

[0041] Figure 13 For the LDA plot of LEfSe analysis.

[0042] Figure 14 For the cladogram plot of LEfSe analysis. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0044] A method for Clostridium butyricum activated feeding of broilers, comprising:

[0045] Clostridium butyricum is activated, inoculated into liquid medium for culture amplification, centrifuged, and the strain is obtained;

[0046] The bacterial strain is pre-frozen at -20 to -18℃ for 4 to 5 hours, then frozen overnight at -80 to -70℃, and then the frozen bacterial strain is pre-cooled to -55 to -57℃, pre-heated for 20 to 25 minutes, main drying for 15 to 18 hours, and final drying for 1 to 1.5 hours to obtain bacterial powder;

[0047] The bacterial powder is subjected to anaerobic liquid fermentation to obtain Clostridium butyricum fermentation liquor;

[0048] The Clostridium butyricum fermentation liquor is added to normal daily diet at 10% (v / w) by wet mixing method, and then fed to obtain the product.

[0049] In some embodiments, the concentration of Clostridium butyricum in the daily diet is 1 to 1.2 x 10 9 CFU / kg of feed.

[0050] In some embodiments, the anaerobic liquid fermentation uses a mechanical stirring anaerobic fermentation system.

[0051] In some embodiments, the use of the fermentation tank includes four steps of empty tank sterilization, filter sterilization, medium sterilization, and inoculation culture.

[0052] In some embodiments, the culture medium used in the fermentation tank culture is a strong Clostridium culture medium.

[0053] In some embodiments, the specific steps of the anaerobic liquid fermentation include three sterilization steps, inoculation of the bacterial powder into the fermentation tank, protection of the fermentation bacteria by nitrogen gas, and culture for 48 to 50 hours.

[0054] In some embodiments, the activation of the bacterial strain is carried out under anaerobic conditions.

[0055] In some embodiments, the Clostridium butyricum is chicken-derived Clostridium butyricum strain FW1, with a biological preservation number of CGMCC NO. 22258.

[0056] The present application will be further described in detail below in conjunction with specific embodiments, it should be pointed out that the specific embodiments are an explanation of the present application rather than a limitation.

[0057] In the following examples, the strong Clostridium culture medium is purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., HB0316.

[0058] Example 1 in vitro activation

[0059] The provided in vitro activation of Clostridium butyricum fermentation agent for improving intestinal health includes the following steps:

[0060] 1. Strain activation: Clostridium butyricum FW1 strain was cultured in the laboratory under anaerobic conditions. The culture medium was a fortified Clostridium culture medium (Qingdao Gaoke High-tech Industrial Park Haibo Biotechnology Co., Ltd., HB0316). The conical flask containing the recovered Clostridium butyricum was placed in an anaerobic bag with an anaerobic bag and color-changing silica gel and incubated in a 37°C incubator under anaerobic conditions for 48 hours. The anaerobic principle was strictly followed. All the above operations were carried out in a sterile clean bench, which met the standard of sterile operating environment.

[0061] 2. Preparation method of Clostridium butyricum powder: 1 mL of Clostridium butyricum (2.7 x 10 5 CFU / mL) cultured and preserved in the laboratory was inoculated into liquid fortified Clostridium culture medium and cultured in an incubator. After 48 hours of culture at 37°C, plate counting was performed, and the data was recorded. Low-speed refrigerated centrifuge was used for centrifugation. The supernatant was discarded after centrifugation, and the strain obtained after centrifugation was placed in a container and pre-frozen for 5 hours at -20°C. Then it was frozen overnight at -80°C. The next day, the frozen strain was placed in a freeze dryer and subjected to four steps of pre-cooling (stabilized at around -56°C), pre-heating (-56°C for 20 minutes), main drying (-56°C for 15 hours), and final drying (-56°C for 1 hour) to obtain the powder (stored at room temperature). 0.05 g of the powder was added to the liquid culture medium for culture, and plate counting was performed after 48 hours. The bacterial content of the powder and the loss of bacteria during freeze-drying were calculated.

[0062] 3. Anaerobic fermentation system scale-up culture (in vitro activation) of Clostridium butyricum: The culture method of Clostridium butyricum in this test was anaerobic liquid fermentation. A 50L mechanical stirring anaerobic fermentation system (SFY-50L mechanical stirring stainless steel fermentation system, Jiangsu Zhenjiang Jianggong Biological Engineering Complete Equipment Co., Ltd.) was used to activate Clostridium butyricum in vitro under anaerobic conditions. The use of the fermentation tank included four steps: sterilization of the empty tank, sterilization of the filter, sterilization of the culture medium, and inoculation and culture. The culture medium used for fermentation tank culture was also a fortified Clostridium culture medium. After three sterilization steps, 1 g of powder (2.9 x 10 7 CFU / mL) was inoculated into the fermentation tank. Nitrogen was introduced to protect the fermentation strain. The culture was incubated at 37°C for 48 hours. When the specified concentration was reached, the bacterial solution was removed and stored at 37±1°C.

[0063] Example 2

[0064] The feed formula was designed according to the feeding standards of broilers at different stages. 10% of the Clostridium butyricum fermentation broth prepared in Example 1 was added to the feed. The feed was uniformly mixed by wet mixing (10%, v / w). The nutritional level and vitamins and trace elements in the diet met the nutritional standards for broilers. The feed formula is shown in Table 1.

[0065] Table 1 Diet composition and nutrient levels of broiler experiment (0-42d) (dry basis, %)

[0066]

[0067]

[0068]

[0069] Note: 1 Vitamin premix (per kg of diet provides): VA, 4.13 mg; pantothenic acid (VB3), 16.67 mg; VE, 60 mg; thiamine (VB1), 1.50 mg; biotin, 0.28 mg; folic acid, 0.87 mg; VD3, 3.00 mg.

[0070] 3.41 mg; VK, 31.5 mg; riboflavin (VB2), 12.50 mg; niacin (VB5), 53.03 mg; pyridoxine (VB6), 6.56 mg;

[0071] Cobalamin (VB12), 1.50 mg; biotin, 0.28 mg; folic acid, 0.87 mg; VD3, 3.00 mg.

[0072] 2 Trace element premix (per kg of diet provides): iodine, 0.92 mg; iron, 518.24 mg; zinc, 278.26 mg; manganese, 376.77 mg; copper, 31.57 mg; selenium, 0.66 mg.

[0073] Trace element premix (per kg of diet provides): iodine, 0.92 mg; iron, 518.24 mg; zinc, 278.26 mg; manganese, 376.77 mg; copper, 31.57 mg; selenium, 0.66 mg.

[0074] 3Actual value; 4Calculated value.

[0075] Example 3

[0076] 1. 360 one-day-old broilers with similar body weight were randomly divided into two groups, with 10 replicates in each group and 18 chickens in each replicate. The two groups of broilers were: feeding the basal diet (CON group) and adding 10% Clostridium butyricum FW1 fermentation broth to the normal diet (FW1 group) (1 x 10 9 CFU / kg of feed). The broilers were raised in 3 layers of cages, with 18 chickens in each cage, and the temperature and humidity in the house were maintained at 23 ± 2°C and 65% ± 3%. The experimental period was 42 days, and sample collection was carried out at 21 and 42 days of age.

[0077] After the start of the experiment, daily feeding after mixing was carried out, and the feed was weighed on the same day. The daily feed intake was calculated, and the body weight change data of each group was recorded once a week. At 21 and 42 days of age, serum was collected after gavage with FITC-dextran reagent for intestinal permeability detection. During the entire experiment, free drinking water was used, and the internal environmental indicators of the chicken house met the standards for livestock and poultry feeding. The internal ventilation of the chicken house was good, and the internal environment of the chicken house was cleaned on time and disinfected regularly. At 21 and 42 days of age, organ index detection was performed, and intestinal segments were collected for intestinal morphology detection, and intestinal mucosa was scraped for intestinal barrier-related gene mRNA expression detection.

[0078] A sterile scalpel was used to cut the cecal contents under sterile conditions. After removal, the sample was immediately placed on ice and labeled in a sterile centrifuge tube. The sample size was 2g per tube to ensure the smooth progress of the experiment. After packaging, the sample was quickly frozen in liquid nitrogen and then transferred to -80°C for storage. To prevent sample contamination, all operations were performed under sterile conditions. The composition of the intestinal microbial community in the cecal contents was analyzed using 16S rRNA sequencing technology. Genomic DNA was extracted from the sample, and the purity and concentration of the DNA were detected. After detection, the V3+V4 variable region was PCR amplified, and the PCR product was purified and recovered. Then the library was constructed, and the constructed library was quantified by Qubit and library detection. After passing the test, the library was sequenced and the data was analyzed.

[0079] 2. Test results

[0080] As shown in Table 2, during the period from 1 to 21 days of age, compared with the CON group, the ADG of the FW1 group was significantly increased (P<0.05), the ADFI was significantly increased (P<0.05), and the FCR had no significant difference (P>0.05); during the period from 21 to 42 days of age, compared with the CON group, the ADG of the FW1 group was significantly increased (P<0.05), the ADFI was significantly decreased (P<0.05), and the FCR was significantly decreased (P<0.05); during the period from 1 to 42 days of age, compared with the CON group, the ADG of the FW1 group was significantly increased (P<0.001), the FCR was significantly decreased (P<0.001), and the average daily feed intake had no significant difference (P>0.05).

[0081] Table 2 Effect of Clostridium butyricum on the production performance of broilers (n=10)

[0082]

[0083] Note: a.b Different letters in each row indicate significant differences (P<0.05).

[0084] As shown in Table 3, at 21 days of age, the FW1 group had significantly lower organ index of liver (P<0.05), bursa of fabricius (P<0.05), leg muscle (P<0.05), and duodenum (P<0.05) than the CON group; there was no significant difference in spleen, thymus, pectoral muscle, femur, tibia, jejunum, and ileum (P>0.05). As shown in Table 4, at 42 days of age, the FW1 group had significantly lower leg muscle index (P<0.05) than the CON group; there was no significant difference in other organ indexes (P>0.05).

[0085] Table 3 Effect of Clostridium butyricum on organ index of 21-day-old broilers (n=10)

[0086]

[0087] Note: a.b Different letters in each row represent significant difference (P<0.05).

[0088] Table 4 Effect of Clostridium butyricum on organ index of 42-day-old broilers (n=10)

[0089]

[0090] Note: a.b Different letters in each row represent significant difference (P<0.05).

[0091] As Figure 1 shown in Table 5, at 21 days of age, there was no significant difference in duodenum villus height, crypt depth, and villus-crypt ratio (P>0.05); at 42 days of age, the FW1 group had significantly higher villus-crypt ratio than the CON group (P<0.05).

[0092] Table 5 Effect of Clostridium butyricum on duodenum morphology of broilers (n=10)

[0093]

[0094] Note: a.b Different letters in each row represent significant difference (P<0.05).

[0095] As Figure 2 shown in Table 6, at 21 days of age, the FW1 group had significantly higher jejunum villus height than the CON group (P<0.05); there was no significant difference in crypt depth and villus-crypt ratio (P>0.05). At 42 days of age, there was no significant difference between the two groups (P>0.05).

[0096] Table 6 Effect of Clostridium butyricum on jejunum morphology of broilers (n=10)

[0097]

[0098] Note: a.b Different letters in each row indicate significant difference (P < 0.05).

[0099] As Figure 3 As shown in Table 7, at 21 days of age, ileal villus height of FW1 group had a significant increasing trend compared with CON group (P = 0.068); at 42 days of age, villus height of FW1 group had a significant increasing trend (P = 0.051), crypt depth had a significant decreasing trend (P = 0.076), and the ratio of villus to crypt had a significant increasing trend (P < 0.001) compared with CON group.

[0100] Table 7 Effects of feeding Clostridium butyricum on the morphology of ileum of broilers (n = 10)

[0101]

[0102] Note: a.b Different letters in each row indicate significant difference (P < 0.05).

[0103] As Figure 4 shown, at 21 days of age, intestinal permeability of FW1 group had a significant increasing trend compared with CON group (P = 0.089); at 42 days of age, there was no significant difference between CON group and FW1 group (P > 0.05).

[0104] As Figure 5 shown, after 21 days of treatment, the expression of MUC2 mRNA in jejunum of FW1 group was significantly up-regulated compared with CON group (P < 0.05); the expression of Occludin mRNA in jejunum of FW1 group had a significant increasing trend (P = 0.054); there was no significant difference in the expression of other genes in jejunum (P > 0.05). There was no significant difference in the expression of intestinal barrier related genes in duodenum and ileum between CON group and FW1 group (P > 0.05).

[0105] As Figure 6 shown, after 42 days of treatment, the expression of MUC5AC mRNA in jejunum of FW1 group was significantly up-regulated compared with CON group (P < 0.05); there was no significant difference in the expression of other genes (P > 0.05). There was no significant difference in the expression of intestinal barrier related genes in duodenum and ileum between CON group and FW1 group (P > 0.05).

[0106] As Figure 7It can be seen that at 21 days of age, the number of OTUs common to both groups was 864, the number of OTUs specific to the CON group was 1498, and the number specific to the FW1 group was 1524. At 42 days of age, the number of OTUs common to both groups was 727, the number of OTUs specific to the CON group was 1532, and the number of OTUs specific to the FW1 group was 2188.

[0107] Table 8 is an analysis of the top 5 species in terms of abundance at the phylum level. As can be seen from Table 8, at 21 days of age, at the phylum level, the abundance of Firmicutes was about 61% in the CON group and about 56% in the FW1 group; the abundance of Bacteroidetes was about 31% in the CON group and about 35% in the FW1 group; the abundance of Proteobacteria was about 6.0% in the CON group and about 7.5% in the FW1 group; the abundance of Tenericutes was about 1.3% in the CON group and about 1.2% in the FW1 group; and the abundance of Actinobacteria was about 0.29% in the CON group and about 0.38% in the FW1 group; none of the above phylum-level abundances were significantly different (P>0.05). At 42 days of age, the abundance of Bacteroidetes was about 54.5% in the CON group and about 60.7% in the FW1 group, and the abundance of Bacteroidetes in the FW1 group had a trend of increasing (P=0.077); the abundance of Proteobacteria was about 2.2% in the CON group and about 3.3% in the FW1 group, which was significantly higher than that in the CON group by 1.1% (P<0.05); and the abundances of the other four phyla at 42 days of age were not significantly different (P>0.05). The phylum-level abundances of the same group at different ages (21 days and 42 days) were compared using two-factor analysis of variance, and the abundance of Firmicutes at 42 days of age was significantly lower than that at 21 days of age (P<0.05), and the abundance of Bacteroidetes was significantly higher (P<0.05). The abundances of the other phyla at 21 days of age and 42 days of age in the same group were not significantly different (P>0.05).

[0108] Table 8 Analysis of the top 5 species in terms of abundance at the phylum level (%)

[0109]

[0110]

[0111] Note: 1 This refers to the use of two-factor analysis of variance by the CON group and the FW1 group to analyze different treatments (normal age and addition of Clostridium butyricum diet) and different periods (21 days of age and 42 days of age).

[0112] a.b Different letters in each row indicate significant differences (P<0.05).

[0113] Table 9 is the analysis of the top 5 species in the genus level. As can be seen in Table 9, at 21 days of age, at the genus level, the abundance of Lactobacillus in the CON group was about 4.8%, and in the FW1 group was about 16.4%; the abundance of Prevotella in the CON group was about 8.9%, and in the FW1 group was about 6.8%; the abundance of Ruminococcus in the CON group was about 7.4%, and in the FW1 group was about 4.8%; the abundance of Bacteroides in the CON group was about 3.1%, and in the FW1 group was about 3.2%; the abundance of Oscillospira in the CON group was about 1.5%, and in the FW1 group was about 1.1%; there was no significant difference (P>0.05). At 42 days of age, there was no significant difference in the abundance of each genus between the CON group and the FW1 group (P>0.05). The genus level bacterial abundance of the same group at 21 days of age and 42 days of age was compared by two-way ANOVA analysis. Compared with 21 days of age, the abundance of Prevotella at 42 days of age was significantly reduced (P<0.05), and the abundance of Oscillospira was significantly increased (P<0.05). There was no significant difference in the abundance of other genera between 21 days of age and 42 days of age (P>0.05).

[0114] Table 9 Analysis of the top 5 species at the genus level (%)(n=6)

[0115]

[0116]

[0117] Note: 1 It means that the CON group and the FW1 group use two-factor analysis method to analyze different treatments (normal age and adding Clostridium butyricum diet) and different periods (21 days of age and 42 days of age).

[0118] a.b Different letters in each row indicate significant difference (P<0.05).

[0119] As Figure 8 At the door level, it can be seen that at 42 days of age, the species abundance distribution of the FW1 group is the most concentrated, the similarity of species distribution is the highest, and the difference between the samples in the group is the smallest. The species distribution of other groups is relatively dispersed, and the difference between the samples in the group is large. Figure 9 At the genus level, at 21 days of age and 42 days of age, the CON group and the FW1 group are densely distributed in the figure, and the difference between the samples in the group is small.

[0120] Alpha diversity analysis was performed, and Figure 10 It can be seen that at 21 days of age, there was no difference in chao 1 index, faith_pd index, observed_features index, simponsion_entropy index and simpson index between the CON group and the FW1 group (P>0.05).

[0121] Depend on Figure 11 It was found that at 42 days of age, there were no differences in the Chao 1 index, observed_features index, simponsion_entropy index, and Simpson index between the CON group and the FW1 group (P>0.05). Compared with the CON group, the faith_pd index of the FW1 group was significantly higher (P<0.05).

[0122] Beta diversity analysis was performed using the PCoA method, and the results are as follows: Figure 12 As shown, at 21 days old, the samples in the CON group and the FW1 group were close in distance and had similar composition. At 42 days old, the samples in the FW1 group were close in distance and had high structural similarity. Compared with the CON group at 42 days old, the samples in the FW1 group were far apart and had different structural composition.

[0123] An analysis of significant differences in OTUs between groups was conducted. Figure 13 and Figure 14 It can be seen that at 21 days of age, the characteristic microorganisms of group CON are Clostridium spp. and Rotiferae; the characteristic microorganisms of group FW1 are Firmicutes, Clostridium spp., Corynebacteriumceae, and Actinomycetes. At 42 days of age, the characteristic microorganisms of group CON are Bacteroidetes, Pasteurella spp., and Clostridium spp.; the characteristic microorganisms of group FW1 are Bacteroidetes, Koala spp., and Veillonellaeaceae.

[0124] Therefore, adding in vitro activated Clostridium butyricum to the diet can improve the intestinal barrier function of broilers, improve intestinal health, increase feed conversion efficiency, improve broiler production performance, and thus increase the economic benefits of broiler farming.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a bacterium agent prepared by an in vitro activation method of broiler chicken Clostridium butyricum in the preparation of a product having any one of the following functions 1) - 3): 1) improving the development of the intestinal tract of broiler chickens, said development of the intestinal tract of broiler chickens comprising: Increased villus height of jejunum and ileum; 2) improving the intestinal barrier function of the broiler chicken, said improving the intestinal barrier function of the broiler chicken comprising: significantly up-regulating the mRNA expression in the jejunum MUC2 mRNA, Occludin mRNA or MUC5AC mRNA expression 3) Optimizing the intestinal flora structure of broiler chicken, which includes: increasing the number of OTUs specific to the flora species, increasing the flora abundance of Bacteroidetes, Proteobacteria or Faecalibacterium prausnitzii, and changing its characteristic flora; The in vitro activation method of broiler chicken Clostridium butyricum comprises the following steps: Activating Clostridium butyricum, inoculating it into a liquid culture medium for culture and amplification, centrifuging to obtain a bacterial strain; The bacterial strain is pre-frozen at -20~-18°C for 4~5 h, then frozen overnight at -80~-70°C, and then the frozen bacterial strain is pre-cooled to -55~-57°C, pre-heated for 20~25 min, main dried for 15~18 h, and finally dried for 1~1.5 h to obtain a bacterial powder; The bacterial powder is subjected to anaerobic liquid fermentation to obtain a Clostridium butyricum fermentation broth; The Clostridium butyricum fermentation broth is added to normal daily ration at 10~15% v / w by wet mixing method, and then fed to obtain the product; The Clostridium butyricum is chicken-derived Clostridium butyricum strain FW1, and the biological preservation number is CGMCC NO. 22258.

2. Use according to claim 1, wherein Dietary C. butyricum concentration was 1-1.2 × 10 9 CFU / kg feed.

3. The use according to claim 1, wherein The anaerobic liquid fermentation uses a mechanical stirring anaerobic fermentation system.

4. The use according to claim 1, wherein The use of the fermentation tank includes four steps of sterilization of the empty tank, sterilization of the filter, sterilization of the culture medium, and inoculation and culture.

5. The use according to claim 1, wherein the compound is ###0002### The culture medium used in the fermentation tank is a strong Clostridium culture medium.

6. The use according to claim 1, wherein The specific steps of the anaerobic liquid fermentation include three sterilization steps, inoculation of the bacterial powder into the fermentation tank, protection of the fermentation bacteria by nitrogen gas, and culture for 48~50 h.

7. The use according to claim 1, wherein the compound is ###0002### The activation of the bacterial strain is carried out under anaerobic conditions.

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