阅读说明：本技术 一种富含酶菌元仔猪替抗饲料复合添加剂及制备方法 (Enzyme-enriched bacterial element-enriched piglet anti-bacterial feed composite additive and preparation method thereof ) 是由 黄遵锡 苗华彪 韩楠玉 唐湘华 张呈波 于 2021-01-21 设计创作，主要内容包括：本发明公开了一种富含酶菌元仔猪替抗饲料复合添加剂及制备方法,该复合添加剂由益生菌、酶制剂和益生元组成。本发明复合添加剂按照500g/t添加到仔猪饲料中,可有效替代仔猪日粮抗生素的使用。添加本发明复合添加剂的仔猪无抗饲料,与基础日粮相比,能够提高仔猪日均采食量和日增重,降低腹泻率和料肉比,生长性能和抗生素组基本一致,且本发明具有良好的经济效益和使用价值。(The invention discloses a piglet anti-bacterial feed composite additive rich in enzyme element and a preparation method thereof. The compound additive is added into piglet feed according to 500g/t, and can effectively replace piglet daily ration antibiotics. Compared with basic ration, the antibiotic-free piglet feed added with the compound additive can improve the daily average feed intake and daily gain of piglets, reduce the diarrhea rate and the feed conversion ratio, and has basically consistent growth performance and antibiotic group, and the antibiotic-free piglet feed has good economic benefit and use value.)

1. The piglet anti-bacterial feed composite additive rich in enzyme bacterial elements is characterized by comprising the following components in parts by weight: 50X 10⁸CFU/g Bacillus subtilis, 50X 10⁸CFU/g bacillus licheniformis, 10000U/g xylanase, 500U/g amylase, 1000U/g glucose oxidase and 15 mass percent of konjac oligomannose.

2. The piglet anti-bacterial feed composite additive rich in enzyme glycogen according to claim 1, wherein the additive amount of the composite additive is 500 g/t.

3. The method for preparing the composite additive of claim 1, comprising the steps of:

1) fermenting bacillus subtilis and bacillus licheniformis to form spores, and performing spray drying to obtain a probiotic semi-finished product;

2) fermenting and drying xylanase, amylase and glucose oxidase to obtain xylanase, amylase and glucose oxidase semi-finished products;

3) carrying out enzymolysis on the konjac fine powder by using beta-mannase to obtain a crude filtrate of konjac mannan oligosaccharide, removing starch, crude fiber particles and residues in oligosaccharide liquid, and carrying out ultrafiltration, concentration and drying to obtain konjac mannan oligosaccharide;

4) and mixing the prepared bacillus subtilis and clostridium butyricum feeding probiotic semi-finished product, feeding xylanase, amylase, glucose oxidase semi-finished product and konjac oligomannose to form the compound feed additive.

Technical Field

The invention belongs to the technical field of feed additives, and particularly relates to a piglet anti-bacterial feed composite additive rich in enzyme bacterial elements and a preparation method thereof.

Background

194 bulletins in the rural agricultural department stop producing commercial feeds containing growth-promoting drug feed additives (except Chinese herbal medicines) from 7 months to 1 day in 2020, so that the breeding industry enters a feed nonreactive age. However, the forbidding of antibiotics brings about the problems of reduction of animal production performance, increase of morbidity, increase of breeding cost, lower benefit and the like in the process of breeding livestock and poultry, and causes great economic loss and technical difficulty for domestic breeding enterprises. How to circumvent the above-mentioned losses in a breeding environment without the use of antibiotics has become an urgent problem to be solved by livestock and poultry breeding.

The products with potential anti-infection potential comprise acidifier, probiotics, prebiotics, enzyme preparation, plant essential oil, fermentation products and the like, and the results of survey reports of the feed society show that: the feed additive probiotics, enzyme preparations and prebiotics substitute antibiotics are 70%, 68% and 53% effective respectively. The microecological preparation is one of the most fire-heat resistant products at present, and has good effects on improving animal intestinal flora, improving animal organism health and improving immunity level, thereby playing a role of 'treating bacteria with bacteria'. The enzyme preparation is used and popularized for many years, the application is very common, and the action mechanism of the enzyme preparation is to supplement the deficiency of endogenous enzyme and improve the digestive activity of the endogenous enzyme; the cell wall of the plant is damaged, and the nutrient digestibility of the feed is improved; the viscosity of intestinal chyme is reduced, and the propagation of harmful microorganisms in the intestinal tract of livestock and poultry is reduced; the intestinal wall structure is changed, the nutrient absorption capacity is improved, and the method is indispensable in antibiotic products. The prebiotics are responsible for selectively changing the composition and metabolism of the intestinal microbial flora, can increase the number of bifidobacteria and other strains which have positive influence on the health of a host, and play a role in regulating the microecology by culturing the endogenous probiotics. However, the solution of the alternative antibiotic problem cannot be simplified, and one product cannot solve all the problems left by the abandonment of antibiotics, so that the combination and the synthesis of the alternative antibiotic product are imperative. The key to successfully replace antibiotics is the effective compatibility of compound medicines, reasonable product collocation and scientific combination scheme.

Disclosure of Invention

The invention aims to provide a piglet anti-bacterial feed composite additive rich in enzyme bacterial elements and a preparation method thereof, and aims to solve the problems of reduced production performance, increased morbidity, increased breeding cost, reduced benefit and the like in the piglet breeding process after antibiotics are forbidden.

In order to achieve the technical purpose, the invention is realized by the following technical scheme:

a piglet anti-bacterial feed composite additive rich in enzyme element comprises probiotics, an enzyme preparation and prebiotics, wherein the probiotics comprises 50 x 10⁸CFU/g Bacillus subtilis CICC 24675 and 50X 10⁸CFU/g Bacillus licheniformis (Bacillus licheniformis CICC 20446); the enzyme preparation comprises: 10000U/g xylanase, 500U/g amylase and 1000U/g glucose oxidase; the prebiotics are konjac oligomannose with the mass fraction of 15%.

The preparation of the probiotics comprises the following steps: activating strains of Bacillus subtilis CICC 24675 and Bacillus licheniformis CICC 20446, transferring the activated strains into a three-stage fermentation culture tank after the strains are subjected to primary and secondary fermentation tank expansion culture, performing fermentation culture for 48 hours, and performing spray drying on a culture solution after the strains form spores of more than 95% to form a semi-finished product of the probiotics for the feeding of the Bacillus subtilis and the Bacillus licheniformis.

Preparation of the enzyme preparation: activating xylanase, amylase and glucose oxidase respectively, performing primary and secondary seed liquid amplification culture, transferring to a three-stage fermentation culture tank when the OD value reaches a certain value, inducing enzyme production by adopting a methanol fed-batch mode, and performing spray drying treatment on the fermentation enzyme liquid when the enzyme activity reaches the maximum value to prepare feed xylanase, amylase and glucose oxidase semi-finished products.

The preparation of the prebiotics comprises the following steps: and (2) carrying out enzymolysis on the konjac fine powder by using beta-mannase, filtering and intercepting the crude fiber after enzymolysis to obtain a crude filtrate of konjac mannan oligosaccharide, removing starch, crude fiber particles and residues in the oligosaccharide liquid, purifying and concentrating by using an ultrafiltration and nanofiltration membrane, and carrying out spray drying by using a spray drying tower to obtain the konjac mannan oligosaccharide.

When the compound additive is used, the compound additive is added into piglet feed according to 500g/t, compared with basic daily ration, the compound additive can improve daily average feed intake and daily gain of piglets, reduce diarrhea rate and feed conversion ratio, has basically consistent growth performance and antibiotic group, and can replace antibiotics for use.

The invention has the beneficial effects that:

the enzyme-bacterium element compound feed additive of the invention with 500g/t is added into piglet feed, which can replace the compound antibiotic in the original feed, thus greatly reducing the problems of environmental pollution and the like caused by antibiotic abuse. And the average daily feed intake and average daily gain of piglets can be effectively improved, the feed conversion ratio and diarrhea rate are reduced, and the breeding benefit is increased.

Drawings

FIG. 1 is a graph of piglet growth performance according to the invention;

FIG. 2 is the rate of diarrhea in piglets according to the invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of feed probiotics

(1) Strain activation

The probiotics used in the examples of the present invention were purchased from the microorganism collection center, Bacillus subtilis cic 24675 and Bacillus licheniformis cic 20446.

LB is adopted as the activation culture medium of the bacillus subtilis and the bacillus licheniformis, and the formula is as follows: 0.5% of yeast extract, 1% of tryptone, 1% of sodium chloride and 2% of agar in a solid culture medium. Inoculating the strain to a plate culture medium, and culturing at 37 ℃ for 24 hours to obtain an activated strain;

(2) fermentation culture

Respectively inoculating the activated strains in the step (1) into liquid LB culture medium to prepare 2L of shake flask seeds, and then transferring the shake flask seeds into 50L and 400L primary and secondary fermentation tanks to culture for 12 hours. Respectively transferring the strains into three-stage 10T fermentation culture media.

The formula of the fermentation medium is as follows: 1% of glucose, 1.5% of corn flour, 1% of yeast powder, 2.5% of soybean meal, 0.45% of sodium citrate, 0.015% of calcium carbonate and 0.002% of magnesium sulfate, culturing for 48 hours at 37 ℃, and canning when the spore rate reaches more than 95%.

(3) Preparation of probiotic semi-finished product

And (3) adding 20% of starch into the fermentation liquor obtained in the step (2), performing spray drying by using a spray drying tower, counting the obtained spray dry powder, and determining the content. The effective viable count of the Bacillus subtilis CICC 24675 can reach 300 multiplied by 10⁸CFU/g, the effective viable count of the Bacillus licheniformis (Bacillus licheniformis CICC 20446) can reach 500 multiplied by 10⁸CFU/g。

Example 2 preparation of a feed enzyme preparation

The enzyme preparation heterologous expression host bacteria used in the embodiment of the invention are constructed in the laboratory.

(1) Seed liquid preparation

A single colony of the pichia pastoris host bacteria serving as the feed enzyme preparation is picked up on an ultra-clean workbench, three-region streaking is carried out on a flat plate, and the strains are activated. After single colonies grow out, selecting the single colonies, inoculating the single colonies in a 200mLYEPD culture medium, culturing 1% of yeast extract, 2% of peptone and 2% of glucose at 30 ℃ and 250rpm for 36-38 hours until the weight of wet cells reaches 30 g/L. Seed amplification culture is carried out by respectively using 50L and 500L fermentation tanks, the formula of a culture medium is 1% of yeast extract, 2% of peptone and 2% of glycerol, the fermentation is carried out for about 24 hours under the conditions of 30 ℃ and 350rpm, 20% (w/v) ammonia solution is added for adjusting the pH value to be approximately equal to 4.5, and the wet cell weight reaches OD600 to be more than 30.

(2) Enzyme-producing fermentation

High-density fermentation medium: each liter of fermentation medium contained 40mL of glycerol, 5.0mL of H₂SO₄And basal salt Medium (26.7mL 85% H₃PO₄、0.93g CaSO₄、18.2g K₂SO₄、14.9g MgSO₄·7H₂O, 4.13g KOH), micronAmount of mineral mixture (6.0g CuSO)₄·5H₂O，0.08g NaI，3.0g MnSO₄·H₂O，0.2g Na₂MoO₄·2H₂O，0.02g H₃BO₃、0.5g CoCl₂、20.0g ZnCl₂、65.0g FeSO₄·7H₂O) and vitamin mixture (128mg C)₆H₁₂O₆、12.8mg C₁₈H₃₂CaN₂O₁₀、12.8mg C₈H₉NO₃·HCl，64mg K₂HPO₄3.2mg of vitamin B1, 0.2g of vitamin B3 and 12.8mg of D-biotin). Fermentation was carried out at 30 ℃ and 50rpm, with pH ≈ 4.7-5.0 controlled by 20% (w/v) ammonia solution, until the wet cell weight reached 64 g/L.

The fermentation process comprises four steps, the first step, glycerol feeding culture step, the whole process is about 17 hours, and pH is controlled until the wet weight reaches 47.2 g/L. In the second stage, after the Dissolved Oxygen (DO) content reached 100% saturation and was maintained for 2 hours, 250L of 50% glycerol was injected into the culture medium at a flow rate of 50L/h, and the process was continued for 5 hours until a wet weight of 140-150 g/L was reached. The dissolved oxygen is maintained at 20% to 30% throughout the process. In the third stage, 250L of a mixture of 50% glycerol and 20% methanol is injected at a rate of 50L/h, and the process is continued for 5h until a wet weight of 140-160 g/L is reached. In the fourth stage, 15L/h of methanol were fed in. The fed-batch fermentation is carried out at 30 ℃ and a pH value of about 4.7-5, meanwhile, ammonia water with 20% (v/v) is added under constant DO (20-30%) for reaction, a DO cascade mode is used, the stirring speed is adjusted to 150-180rpm, the inlet air is 1-1.2 VVM, and the physical pressure of the whole fed-batch culture stage is kept at 0.05 MPa. This stage lasts 120 to 150 hours.

(3) Preparation of feed enzyme preparation semi-finished product

And (3) adding 20% of starch into the fermentation liquor obtained in the step (2), performing spray drying by using a spray drying tower, and performing enzyme activity determination on the obtained spray dry powder. The xylanase enzyme activity is 100000U/g, the amylase enzyme activity is 5000U/g and the glucose oxidase enzyme activity is 5000U/g.

EXAMPLE 3 preparation of oligomannooligosaccharides

(1) Preparation of hydrolysate of konjac oligosaccharide

Hydrolyzing 25% rhizoma Amorphophalli refined powder with beta-mannase in an enzymolysis tank at 60 deg.C and 50IU/mL, stirring for hydrolyzing for 1 hr, heating to 80 deg.C, maintaining for 10min, stopping enzymolysis, cooling, and filtering and retaining crude fiber in the hydrolysate with a screen.

(2) Filtering and concentrating

Firstly, removing starch, coarse fiber particles and residues in the oligose liquid by high-speed centrifugation at 8000rpm, intercepting a small amount of particulate matters such as protein, soluble starch, fibers and the like in the oligose liquid by using a ceramic membrane with the pore diameter of 0.2 mu m, setting the pressure of a pipeline to be 0.3MPa, and continuously and circularly washing and filtering the oligose liquid by adding water in the middle until the sugar degree of the filtered liquid is lower than 1 DEG Bx. And finally, adding the oligosaccharide solution filtered by the ceramic membrane into ultrafiltration membrane equipment, setting the filtering pressure to be 0.3MPa, and starting filtering and separating. And pouring the ultrafiltrate into nanofiltration buffer equipment, setting the concentration pressure to be 1.5MPa, starting the equipment, and starting concentration.

(3) Spray drying

And (3) performing spray drying on the konjac oligosaccharide solution purified and concentrated by the nanofiltration membrane by using a spray drying tower. Starting a draught fan of the drying tower, starting a heating device of the drying tower to heat the interior of the drying tower, turning on an atomizer device when the temperature in the drying tower rises to 180 ℃, turning on a feeding pump power supply with the rotation speed of 2000r/min, setting the flow speed of a peristaltic pump to be 5L/h, and starting the peristaltic pump to start oligosaccharide liquid spray drying.

EXAMPLE 4 preparation of enzyme-Yeast composite feed additive

An enzyme-yeast composite feed additive was prepared by using the probiotic bacteria for feed of example 1, the enzyme preparation for feed of example 2, and the konjac mannan oligosaccharide semi-finished product for feed of example 3, and contained 166.7g of Bacillus subtilis (cic 24675), 100g of Bacillus licheniformis (cic 20446), 100g of xylanase, 100g of amylase, 200g of glucose oxidase, 150g of konjac mannan oligosaccharide, and 183.3g of corn starch per kg, and they were mixed uniformly by using a mixer. The final content of Bacillus subtilis CICC 24675 is 50 × 10⁸CFU/g and lichenThe content of Bacillus (Bacillus licheniformis CICC 20446) is 50 × 10⁸CFU/g, xylanase enzyme activity of 10000U/g, amylase enzyme activity of 500U/g, glucose oxidase enzyme activity of 1000U/g, and konjac oligomannose content of 15%.

Example 5 piglet cultivation experiment

1) Preparation of piglet feed

The enzyme-bacterium element composite feed additive is added into basal ration at a ratio of 500g/t for replacing antibiotic feed for piglets, the positive control group is formed by adding composite antibiotic into antibiotic feed on the basis of the basal ration, and the negative control group is formed by feeding the basal ration. The three types of daily ration are mixed evenly by a mixer and granulated to form 3 different piglet daily rations. The nutritional levels of crude protein, calcium, total phosphorus and the like in the daily ration of each group are consistent, and the nutritional levels refer to NRC (2012) and Chinese pig feeding standards (2004). The recipe of each treatment is shown in table 1, and the nutritional level of the recipe is shown in table 2.

Table 1 recipe (%)

TABLE 2 formula nutrient index

2) Experimental protocol

Adopting a totally enclosed pigsty, automatically controlling the temperature, the humidity, the ventilation intensity and the carbon dioxide and ammonia concentration in the pigsty, keeping the pigsty temperature at 24-26 ℃ during the test period, feeding the test piglets in different columns, and feeding the test piglets at 1.5 multiplied by 1.5m²In the ring, a plastic-sprayed floor with a leak, a stainless steel adjustable trough and a nipple-shaped water fountain. The granular feed is used for feeding, and the feed can be freely taken and drunk. At the beginning of the experiment, at 1 day and 28 days, the individual weight of the weaned piglets and the feed weight are respectively weighed, and the Average Daily Gain (ADG), the Average Daily Feed Intake (ADFI) and the feed-weight ratio (feed: gain, F/G) are calculated. The weight of the dead-panned pigs and the weight of this duplicate remaining material were recorded at the time of dead panning of the pigs and used to correct growth performance data.

Piglets were recorded for diarrhea at a fixed point twice a day (morning, afternoon) and diarrhea rate and diarrhea score were calculated for each treatment group.

Normal, cylindrical (0 min);

mild diarrhea: thin, soft, tangible (1 point);

moderate diarrhea: sticky, unshaped, high water content (2 min);

severe diarrhea: liquid, unformed, watery manure (3 min).

Among them, 2 points (including 2 points) are marked as diarrhea, and the treatment condition is recorded in detail.

Average Daily Gain (ADG) ═ average daily gain (ADFI) ((end of test weight-initial test weight)/day of test) (feed in test period-feed remaining in test period)/day of test; feed Conversion Ratio (FCR) average daily feed intake/average daily gain; diarrhea rate ═ diarrhea pig heads/(test pig heads total days) ×; diarrhea score-total stool score/(number of test pigs total days).

The experimental results are as follows: as can be seen from fig. 1, compared with the non-resistant diet group, piglets fed with 500g/t of the enzyme-bacterium composite feed additive plus the basic diet of the invention can reduce the feed-meat ratio by 4.52%, and the difference is significant (P < 0.05); the daily gain is increased by 11.46 percent, the daily average feed intake is increased by 6.73 percent, and the difference is obvious (P is less than 0.05); compared with a positive control group with anti-daily ration, the feed-meat ratio difference is not significant (P is greater than 0.05), the daily average feed intake and the daily gain are respectively improved by 5.32 percent and 4.39 percent, and the difference is significant (P is less than 0.05). As can be seen from fig. 2, the diarrhea rate of the alternative antibody regimen was reduced by 61.54% compared to the negative control group, and the difference was very significant (P < 0.01).

In conclusion, the invention provides the composite microecological feed additive capable of replacing antibiotics in the piglet daily feed through reasonable optimization and compatibility and the combination of probiotics, the composite microecological feed additive can increase daily feed intake and daily weight gain in the piglet breeding process, reduce feed conversion ratio and diarrhea rate, thereby increasing production performance and experience benefits in the piglet breeding process and simultaneously playing a role in environmental protection.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.