阅读说明：本技术 一种产淀粉酶和木聚糖酶的蜡样芽孢杆菌在禽类发酵饲料中的应用 (Application of bacillus cereus for producing amylase and xylanase in poultry fermented feed ) 是由 冯焱 王雷 郝琴琴 冯江浩 姚军虎 于 2020-08-11 设计创作，主要内容包括：本发明提供了一种产淀粉酶和木聚糖酶的蜡样芽孢杆菌菌株Bacillus cereus W3。本发明还公开了一种用于改善家禽纤维消化能力的饲用微生物添加剂,由高纤维饲料原料为基质,对蜡样芽孢杆菌菌株Bacillus cereus W3进行富集发酵制备而成,活菌发酵量1013CFU/g时,在玉米-豆粕型饲粮中添加5％饲用微生物添加剂替代玉米,使用效果显著。本发明能提高家禽肠道中淀粉酶和木聚糖酶的活性,优化肠道菌群结构,增强家禽对高纤维饲料的消化吸收能力,对提高家禽饲料转化效率和新型饲料开发与高效利用具有指导意义。(The invention provides a Bacillus cereus strain Bacillus cereus W3 for producing amylase and xylanase. The invention also discloses a feeding microbial additive for improving the fiber digestibility of poultry, which is prepared by taking a high-fiber feed raw material as a substrate and carrying out enrichment fermentation on the Bacillus cereus strain Bacillus cereus W3, wherein when the viable bacteria fermentation amount is 1013CFU/g, 5 percent of the feeding microbial additive is added into the corn-soybean meal type feed to replace corn, and the using effect is obvious. The invention can improve the activity of amylase and xylanase in poultry intestines, optimize the intestinal flora structure, enhance the digestion and absorption capacity of poultry on high fiber feed, and has guiding significance for improving the poultry feed conversion efficiency and developing and efficiently utilizing novel feed.)

1. The application of the agricultural byproduct wheat bran fermentation product in preparing poultry feed is characterized in that the agricultural byproduct wheat bran fermentation product is prepared by adopting the following method:

s1, cleaning and sterilizing wheat bran, and adding a fermentation buffer solution;

s2, activating and culturing a Bacillus cereus W3 strain to obtain activated bacterial liquid; then adding the mixture into S1 for fermentation to obtain a fermentation product;

in the S1, the wheat bran accounts for 5-8% of the mass of the fermentation buffer solution, and the pH value of the fermentation buffer solution is 6.8-7.5; in S2, a Bacillus cereus W3 for producing xylanase and amylase is preserved in China center for type culture Collection (CGMCC) No.19785 in 5-8.2020.

2. A feeding microbial additive for improving the fiber digestibility of poultry prepared by Bacillus cereus W3 according to claim 1, wherein: it comprises two functional components of Bacillus cereus W3 and a fermentation substrate.

3. The microbial feed additive for improving the digestibility of poultry fibers according to claim 2, wherein the fermentation substrate is selected from common poultry high fiber feed stocks, particularly wheat bran.

4. The feed microbial additive for improving the fiber digestibility of poultry according to any one of claims 2 and 3, wherein the fermentation temperature for preparing the feed microbial additive is 28-45 ℃, the water content is 10-40%, and the final viable bacteria fermentation amount is 10%¹¹-10¹⁶CFU/g。

5. The microbial feed additive for improving the fiber digestibility of poultry according to any one of claims 2, 3 and 4, wherein the microbial feed additive is added in an amount of 3 to 25 percent in the corn-soybean meal feed.

6. The use of Bacillus cereus W3 as claimed in any one of claims 1, 2, 3, 4 and 5 for improving the fiber digestibility of poultry in poultry farming and the use of the microbial feed additive prepared therefrom.

Technical Field

The invention relates to a Bacillus cereus W3 for producing amylase and xylanase and application thereof in preparation of a microbial feed additive, belonging to the technical field of breeding feed.

Background

China is the first agricultural kingdom of the world poultry meat yield, and particularly intensive breeding greatly reduces the production cost, so that the poultry meat becomes a high-quality animal protein source with rich nutrition and substantial price. The feed conversion efficiency of poultry is higher compared with that of pigs and ruminants, but because the poultry can not secrete cellulase systems, the poultry feed has weak digestion capability on feed raw materials with higher fiber content such as wheat, wheat bran and the like, and thus the poultry feed becomes a main bottleneck for limiting the selection and the high-efficiency utilization of the raw materials of the poultry feed.

Bacillus cereus (Bacillus cereus) can be subjected to enrichment culture through solid state fermentation, and part of strains can secrete amylase and xylanase simultaneously. Amylase is one of the main endogenous enzymes of animals and plays an important role in feed digestion; the xylanase can degrade xylan in a hemicellulose structure in the feed, and the produced xylooligosaccharide can be utilized by microorganisms or animals to avoid nutrition loss, so that the conversion efficiency of poultry on high-fiber feed nutrition is integrally improved. In addition, Bacillus cereus as probiotic bacteria can regulate the structure of intestinal flora, inhibit the propagation of harmful flora, reduce digestive tract diseases and promote intestinal health. Therefore, Bacillus cereus can be used as a microbial additive to be applied to the feed industry.

Disclosure of Invention

The invention aims to release more nutrients by microbial fermentation, which is difficult to directly utilize the nutrients in wheat bran, so as to improve the production performance of poultry.

In order to solve the problem of insufficient digestive ability of poultry on fiber feed and make up the defect of fiber nutrition loss in the feed caused by insufficient digestive tract secretion enzyme system, the invention provides a Bacillus cereus W3 for producing amylase and xylanase and a feeding microbial additive prepared from the Bacillus cereus W3 for improving the fiber digestive ability of poultry.

The technical scheme adopted by the invention for solving the problems is as follows:

provides a Bacillus cereus W3 for producing amylase and xylanase, and the preservation number is CGMCC No. 19785.

The Bacillus cereus W3 is used for preparing a feeding microbial additive for improving the fiber digestibility of poultry, which comprises two functional components of Bacillus cereus W3 and a fermentation substrate. The microbial feed additive can be applied to poultry feeds, and is mainly used for improving the digestibility of poultry on fiber feeds, improving the intestinal flora structure of poultry and improving the conversion efficiency of fiber feeds.

The fermentation substrate of the microbial additive for feeding can be selected from common high-fiber feed raw materials for poultry, and wheat bran is preferred.

The fermentation temperature of the microbial additive for feed is 28-45 ℃, the water content is 10-40%, and the final viable bacteria fermentation amount is 10%¹¹-10¹⁶CFU/g, preferably fermentation temperature of 35-37 deg.C, water content of 15% -20%, and final viable bacteria fermentation amount of 10%¹³-10¹⁴CFU/g。

The addition amount of the feeding microbial additive in the corn-soybean meal type feed is 3-25%, preferably 5-15%.

The Bacillus cereus W3 and the feed microbial additive prepared by the same for improving the fiber digestibility of poultry have the advantages that the activities of digestive enzymes of poultry intestinal tracts, particularly amylase and xylanase can be improved, the defect of the secretory capacity of a poultry cellulase system is compensated, and the digestibility of the poultry on high-fiber feed is improved; meanwhile, the structure of intestinal flora is optimized, the propagation of harmful bacteria is reduced, and the overall conversion efficiency of the poultry feed is improved.

Biological preservation

The bacillus cereus is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of Beijing city Kogyo Kyoho, Microbiol research institute of Chinese academy of sciences, postal code: 100101) at 5, 8 and 5 months in 2020 with the preservation number of CGMCC No. 19785.

Drawings

FIG. 1 is a line graph of amylase and xylanase enzymes from Bacillus cereus W3 at different fermentation times according to the present invention;

FIG. 2 is a PCoA analysis result of the effect of the feeding microbial additive on the intestinal microbial structure of the broiler chicken;

wherein the fitting process adopts an Unweighted UniFrac distance algorithm;

wherein the ellipses represent fitting areas of the intestinal microbial structures of the same processing group samples, and the more obvious the separation among the ellipses, the more obvious the intestinal microbial structure differentiation among the corresponding processing group samples;

wherein the R value and the P value are the results of difference replacement test among ANOSIM groups, and the replacement times are 999 times.

Detailed Description

The present invention will be more readily understood with reference to the following specific examples. The specific examples, animal, feed formulation and manufacturing processes and results described in the examples are illustrative of the invention and are not to be construed as limiting the invention. The examples relate to animals, feed materials and reagents and are commercially available without specific reference.

Example 1 determination of liquid fermentation Amylase and xylanase by Bacillus cereus W3

(1) Liquid fermentation and enzyme liquid extraction

Taking 100 mu L of Bacillus cereus W3 glycerobacteria, inoculating into 30mL LB culture medium, oscillating at 200r/min and 37 ℃, fermenting and culturing, sucking 1mL of bacterial liquid after 0, 24, 48, 72, 96 and 120h respectively, centrifuging at 4 ℃ and 6000r/min for 1min to remove bacteria, sucking supernatant, and transferring to 4 ℃ precooled sterile EP to be used as enzyme liquid.

(2) Amylase and xylanase Activity assays

The substrates for the enzymatic reaction are as follows:

starch: Tris-HCl buffer (0.1mol/L, pH 8.0) solution of 1g/mL starch 400. mu.L.

Xylan: 0.2g/mL xylan in sodium citrate buffer (0.1mol/L, pH 5.0) 400. mu.L.

Sequentially adding 30 mu L of enzyme solution into a centrifuge tube containing the substrates, and carrying out water bath reaction at 55 ℃ for 30 min; the control group was mixed, reacted at 100 ℃ for 10min to inactivate the xylanase, and subjected to a 55 ℃ water bath. And then 860 mu L of DNS reagent is mixed into 430 mu L of enzymatic reaction system, after the reaction is carried out for 10min by heating in a metal bath at 100 ℃, 1mL of reaction solution is absorbed and diluted by 4mL of deionized water, a control group is used for marking zero, the absorbance value of the diluted reaction solution at 540nm is measured, the generation amount of reducing sugar is calculated by contrasting a reducing sugar-OD 540 standard curve, and the activity of amylase and xylanase is judged.

Definition of enzyme activity unit: under the above reaction conditions, the amount of enzyme required for 1mL of the fermentation product per minute to degrade from a sufficient amount of substrate and release 1. mu. mol of reducing sugar is one unit of enzyme activity, U/mL. The calculation formula is as follows:

EA＝S×D×1000/(V×T×M)

in the formula: EA-enzyme activity of fermentation sample, U/mL; production of S-reducing sugar, mg; d, dilution times of enzyme solution; 1000-molar conversion coefficient, 1mmol ═ 1000 μmol; v-the addition amount of enzyme, mL; t-reaction time, min; relative molecular mass of the M-reducing sugar, g/mol.

Test results and analysis

The activities of amylase and xylanase of the Bacillus cereus W3 in different fermentation time are respectively calculated according to a formula, and a fermentation enzyme activity curve is drawn, and is shown in figure 1. The activities of the amylase and the xylanase of the bacillus cereus W3 are highest at the same time after 96-hour fermentation, and are 49.78U/mL and 56.91U/mL respectively.

Example 2 Effect of Bacillus cereus W3 preparation of feed microbial preparation on broiler chickens

(1) Fermentation preparation of microbial additives

Performing solid state fermentation with Bacillus cereus W3, supplementing water to water content of 20% with wheat bran as nutrient medium, adjusting pH to natural, and standingStirring and fermenting in a constant-temperature fermentation tank at 37 ℃ until the fermentation quantity of viable bacteria is 10¹³CFU/g。

(2) Test animals and groups

Selecting 180 feathers of 21-day-old white feather broilers, randomly dividing the white feather broilers into 3 groups, and repeating the groups by 6, wherein 10 feathers of broilers are repeated. The feed formulations for the different groups are shown in table 1. The feed raw materials are weighed and mixed according to the feed formula, and are crushed, mixed and granulated. The microbial feed additives in the fermented feed raw materials are prepared in batches every 5 days to ensure freshness and effectiveness, and the rest substances are premixed in advance, mixed with the microbial feed additives in proportion, fully mixed and granulated.

Control group: corn-soybean meal type feeds;

wheat bran group: 5 percent of wheat bran is used for replacing corns in the corn-soybean meal type feed, and other nutrient components are micro-adjusted to keep the metabolic energy of the poultry unchanged;

and (3) fermentation group: the corn-soybean meal type feed is replaced by 5 percent of feeding microbial additive (the fermentation amount of viable bacteria is 10)¹³CFU/g), and micro-adjusting other nutrient components to keep the metabolism of the poultry unchanged.

TABLE 1 Dry-based feed formulation and nutrient content for different treatment groups

¹The premix minerals provide the following nutrients per kg of feed: cu (copper sulfate) 8 mg; 80mg of Fe (ferrous sulfate); zn (zinc sulfate) 80 mg; mn (manganese sulfate) 100 mg; i (calcium iodide) 0.35 mg; se (sodium selenite) 0.30 mg.

²The poultry vitamins provide the following nutrients for each kilogram of feed: retinol 18000 IU; cholecalciferol 2000 IU; 20IU of tocopherol; vitamin K0.5 mg; thiamine 2 g; 8mg of riboflavin; 10mg of nicotinic acid; 35mg of pantothenic acid; pyridoxine 3.5 mg; biotin 0.18 mg; folic acid 0.55 mg; cobalamin 0.01 mg.

(3) Feeding management

The feeding test period is 21-42 days old, three layers of cage culture are adopted, the environment humidity is kept at 50%, the temperature of the henhouse is 26 ℃ when the henhouse is 21 days old, the temperature is reduced by 0.3 ℃ every day later, and ventilation is kept. Each group supplies enough corresponding feed, and water drinking and food intake are free in the feeding process.

(4) Measurement index

Average daily gain, feed-to-weight ratio, intestinal enzyme activity, and microbial diversity.

(5) Results and analysis

The results of the effects of the microbial additive on broiler chicken production, intestinal enzyme activity and intestinal microbial diversity are shown in table 2 and figure 2. After the microbial additive is added into a fermentation group, the daily gain and the feed-weight ratio of broiler chickens are obviously improved, the activities of amylase and xylanase in duodenum are respectively improved by 227% and 62% compared with a control group, the microbe abundance of cecum is obviously improved, and the flora structure is also obviously improved. In conclusion, the microbial additive provided by the invention has good effects on improving the digestibility of broiler feed, improving the intestinal flora structure and improving the production efficiency.

TABLE 2 influence of wheat bran fermentation microbial additives on broiler growth and intestinal tract