Solid-state bacterium-enzyme synergistic fermented feed as well as preparation method and application thereof

Abstract

The invention provides a solid-state bacterium-enzyme synergistic fermented feed as well as a preparation method and application thereof. By establishing and optimizing a synergistic fermentation process of complex microbial inoculants such as bacillus subtilis, lactobacillus plantarum, saccharomyces cerevisiae, aspergillus niger and the like and nine types of enzyme preparations, it is found that the fermentation can significantly improve the content of nutritional ingredients of the feed and effectively degrade toxins and anti-nutritional factors. 16S rRNA gene sequencing and flavor omics sequencing find that the fermentation can also significantly improve the abundance of microorganisms such as lactobacillus and pediococcus in the feed, and produced flavor substances endow the feed with outstanding sweet, fruity, green grass and waxy taste characteristics. In addition, through further observation of feeding growing pigs, the feed can significantly improve the growth performance of the pigs, enhance the oxidation resistance of the body, and effectively improve the small intestine tissue morphology and the intestinal barrier function. The feed effectively solves the problems of slow growth and high incidence of intestinal diseases of young pigs, and provides key technical support for antibiotic-free breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture feed, and in particular to a solid-state bacterial enzyme synergistic fermentation feed and a preparation method and application thereof. Background Art

[0002] The primary feed type used in my country's livestock and poultry industry is corn-soybean meal. While corn and soybean meal are high-quality nutritional sources in pig diets, they still contain anti-nutritional factors such as soybean antigen proteins, trypsin inhibitors, and non-starch polysaccharides. These factors restrict the release and absorption of nutrients and can cause intestinal allergies and diarrhea in pigs. Furthermore, the intestinal tract of young animals is still underdeveloped. Since the advent of the antibiotic-free feeding era, particularly the ban on growth-promoting antibiotic additives, the livestock industry has faced significant challenges in ensuring the growth and health of young animals.

[0003] Fermented feed can effectively reduce anti-nutritional factors in feed, reduce intestinal immunogenic reactions, and improve the production performance of pigs at all stages. At present, fermented feed is mostly concentrated in the application of liquid fermentation, but liquid fermented feed is not easy to store, the range of strains used is mostly limited to bacteria, and the preparation process is cumbersome. Solid-state fermentation is applicable to a wide range of strains, has a large amount of fermentation base, and a simple operation process. In addition, the additional addition of enzyme preparations to assist strain fermentation can overcome the problems of unstable strain performance and low enzyme production, and help reduce feed quality losses caused by fermentation. This advantage plays a key role in the development and utilization of animal feed sources.

[0004] However, there is relatively little research on the solid-state fermentation of bacteria, fungi and enzyme preparations involved in solid-state bacterial-enzyme synergistic fermentation, and the preparation methods and applications of fermented feed need to be comprehensively and systematically studied. Summary of the Invention

[0005] The purpose of the present invention is to provide a solid-state bacterial enzyme synergistic fermentation feed and a preparation method and application thereof.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a bacterial enzyme composite microecological preparation, which is prepared by mixing a composite bacterial agent and a composite enzyme preparation in proportion; The composite bacterial agent comprises Bacillus subtilis ( Bacillus subtilis )、Lactobacillus plantarum( Lactobacillus plantarum ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Aspergillus niger ( Aspergillus niger ); The complex enzyme preparation comprises alpha-amylase, lipase, protease, cellulase, pectinase, xylanase, alpha-galactosidase, beta-glucanase and glucose oxidase.

[0007] Furthermore, the bacterial enzyme composite microecological preparation is prepared by mixing the composite bacterial agent and the composite enzyme preparation in a ratio of 9 mL:3.49 g.

[0008] Furthermore, the composite bacterial agent is prepared by mixing Bacillus subtilis liquid, Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Aspergillus niger liquid in a volume ratio of 1:2:3:3; wherein the bacterial content of the Lactobacillus plantarum liquid is 4.0×10 9 ~4.5×10 9 CFU / mL, the bacterial content of Bacillus subtilis liquid was 1.0×10 8 ~1.5×10 8 CFU / mL, the bacterial content of the Saccharomyces cerevisiae liquid was 4.0×10 7 ~4.5×10 7 CFU / mL, the bacterial content of Aspergillus niger liquid is 1×10 6 CFU / mL~2×10 6 CFU / mL.

[0009] Furthermore, the contents of various enzymes in the solid composite enzyme preparation are 3000U / g of α-amylase, 10000U / g of lipase, 60000U / g of protease, 10000U / g of cellulase, 30000U / g of pectinase, 1000000U / g of xylanase, 500U / g of α-galactosidase, 50000U / g of β-glucanase and 10000U / g of glucose oxidase.

[0010] Furthermore, the accession number of the Lactobacillus plantarum is BNCC336421, the accession number of the Bacillus subtilis is BNCC109047, the accession number of the Saccharomyces cerevisiae is CICC1355, and the accession number of the Aspergillus niger is CICC40273.

[0011] Furthermore, the EC numbers of the enzymes in the complex enzyme preparation are: α-amylase EC 3.2.1.1, lipase EC 3.1.1.3, protease EC 3.4.22.2, cellulase EC 3.2.1.4, pectinase EC 3.2.1.15, xylanase EC3.2.1.8, α-galactosidase EC 3.2.1.22, β-glucanase EC 3.2.1.73, and glucose oxidase EC 1.1.3.4.

[0012] In a second aspect, the present invention provides the application of the bacterial enzyme composite microecological preparation in the field of feed processing.

[0013] In a third aspect, the present invention provides a solid-state bacterial enzyme synergistic fermentation feed, which is obtained by mixing and fermenting the bacterial enzyme composite microecological preparation with feed (preferably pig feed, such as corn-soybean meal feed).

[0014] Preferably, the fermentation method is solid-state fermentation.

[0015] Furthermore, the amount of the bacterial enzyme compound probiotic preparation added to the corn-soybean meal type feed is 9 mL (ie, 9 mL of the bacterial enzyme compound probiotic preparation is added to every 100 g of corn-soybean meal type feed).

[0016] Preferably, the corn-soybean meal feed includes the following components in grams: 67.46g corn, 3g whey powder, 1.23g soybean oil, 20.61g soybean meal, 2g fish meal, 1.57g calcium hydrogen phosphate, 0.48g stone powder, 0.56g sodium chloride, 0.55g lysine phosphate, 0.23g methionine, 0.23g threonine, 0.08g tryptophan and 2g premix.

[0017] Furthermore, the premix provides, per kilogram of feed, 10,000 IU of vitamin A, 3,500 IU of vitamin D, 70 IU of vitamin E, 3 mg of vitamin K3, 3 mg of vitamin B1, 10 mg of vitamin B2, 6 mg of vitamin B6, 0.4 mg of biotin, 30 mg of pantothenic acid, 30 mg of niacin, 110 mg of copper (copper sulfate pentahydrate), 10 mg of manganese (manganese sulfate), 140 mg of iron (ferrous sulfate), 0.5 mg of iodine (potassium iodide), and 0.3 mg of selenium (sodium selenite).

[0018] In a fourth aspect, the present invention provides a method for preparing the feed, comprising uniformly mixing the bacterial enzyme composite microecological preparation and corn-soybean meal feed in proportion, adding water to adjust the moisture content to 72%, and fermenting the mixture under sealed conditions at 36°C for 4.1 days.

[0019] In a fifth aspect, the present invention provides any of the following applications of the preparation method: 1) Used to increase the content of ash (Ash), acid-soluble protein (TCA-SP), OM (organic matter), CP (crude protein) and EE (crude fat) in corn-soybean meal feed, thereby improving the nutritional value of the feed; 2) Used to reduce the content of anti-nutritional factors and toxins in corn-soybean meal feed; 3) Used to increase the abundance of beneficial bacteria in corn-soybean meal feed; 4) Used to increase the content of flavor compounds such as aldehydes, acids, esters and phenols in fermented corn-soybean meal feed, making the feed have a sweet, fruity, grassy and waxy taste, and improving the palatability of the feed.

[0020] Preferably, the anti-nutritional factors in 2) include but are not limited to α-conglycinin, β-conglycinin, soybean antigen protein, trypsin inhibitor TI and lectin, and the toxins include but are not limited to DON and ZEN.

[0021] Preferably, the beneficial bacteria in 3) include but are not limited to Lactobacillus ( Lactobacillus ) and Pediococcus ( Pediococcus )microorganism.

[0022] In a sixth aspect, the present invention provides any one of the following applications of the feed: (1) Used in pig farming; (2) Used to increase the average daily weight gain of young pigs and reduce feed conversion rate; (3) Used to increase the villus height, crypt depth and villus-to-crypt ratio of the small intestine of growing pigs; (4) Used to increase the expression of pig intestinal tight junction proteins Claudin-1, Occludin and ZO-1.

[0023] Furthermore, (1) the feed is used to replace 10-15% (preferably 10%) of the complete feed for feeding.

[0024] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (1) This invention establishes a highly efficient solid-state fermentation process that combines bacteria and enzymes: This invention innovatively combines a specific composite bacterial strain (Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus niger, with an optimal ratio of 1:2:3:3) with a specific composite enzyme system (containing nine enzyme preparations, including α-amylase) for solid-state fermentation of complete pig feed (optimized conditions: temperature 36°C, inoculation volume 9.3%, moisture content 72%, and fermentation time 4.1 days). The bacterial strains and enzyme preparations work synergistically, and the small molecules produced by microbial fermentation promote enzymatic hydrolysis efficiency. At the same time, the enzymatic hydrolysis products accelerate microbial growth and metabolism, overcoming the problems of insufficient enzyme production or incomplete enzymatic hydrolysis caused by individual bacterial fermentation, significantly improving fermentation efficiency.

[0025] (II) Significantly improve feed quality: effectively increase the nutrient content of feed (such as crude protein); significantly degrade anti-nutritional factors (ANFs) and mycotoxins in feed; based on 16S rRNA gene sequencing analysis, it was found that the fermentation process significantly increased the Lactobacillus genus ( Lactobacillus ) and Pediococcus ( Pediococcus ) and other beneficial microorganisms, optimizing the microbial composition of the feed; flavor omics analysis confirmed that the fermentation process produced unique changes in flavor substances, giving the feed prominent sweet, fruity, grassy and waxy flavor characteristics, thereby improving the palatability of the feed.

[0026] (3) Effectively promote the health and growth of pigs: Feeding the solid-state bacterial enzyme synergistically fermented feed prepared by the present invention (replacing 10-15% of complete feed) can significantly improve the growth performance of growing pigs, enhance the body's antioxidant capacity, and effectively improve the small intestinal tissue morphology and intestinal barrier function, ultimately promoting the healthy growth of pigs.

[0027] (4) Easy operation and strong practicality: Directly using complete pig feed for solid-state fermentation eliminates the step of preparing complex fermentation substrates. Compared with liquid fermentation, it is simpler and less costly, and easier to promote and apply in actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the effect of mixed bacteria fermentation in different proportions on the resistance of nutritional factors in a preferred embodiment of the present invention.

[0029] Figure 2 This is the effect of different fermentation conditions on solid-state fermentation feed in a preferred embodiment of the present invention.

[0030] Figure 3 This is a response surface optimization experiment on solid-state fermentation feed under different fermentation conditions in a preferred embodiment of the present invention.

[0031] Figure 4 This is the bacterial enzyme synergistic fermentation effect in a preferred embodiment of the present invention.

[0032] Figure 5 This is the surface structure and protein molecular weight changes of the bacterial enzyme solid-state fermentation feed in the preferred embodiment of the present invention.

[0033] Figure 6 It is the Alpha diversity index of the solid-state fermentation feed in a preferred embodiment of the present invention.

[0034] Figure 7 This is the analysis result of the microbial structure of solid-state fermentation feed in a preferred embodiment of the present invention.

[0035] Figure 8 This is a cumulative diagram of the relative abundance of the phylum and genus levels of solid-state fermented feed in a preferred embodiment of the present invention.

[0036] Figure 9 This is the result of multi-level species difference discrimination analysis of solid-state fermented feed LEfSe in a preferred embodiment of the present invention.

[0037] Figure 10 This is a river diagram of the functional abundance of solid-state fermentation feed microorganisms in a preferred embodiment of the present invention.

[0038] Figure 11 The following is an identification and annotation of flavor substances in a preferred embodiment of the present invention.

[0039] Figure 12The relative odor activity value and sensory flavor radar chart in the preferred embodiment of the present invention.

[0040] Figure 13 This is a volcano plot of flavor metabolites in a preferred embodiment of the present invention.

[0041] Figure 14 This is a network diagram of the association between sensory flavor characteristics and flavor substances in a preferred embodiment of the present invention.

[0042] Figure 15 This is the effect of solid-state fermentation feed on small intestinal barrier function in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention aims to provide a solid-state bacterial enzyme-co-fermented feed for pigs, as well as its preparation method and application. This method can effectively enhance the nutritional value of the feed, degrade anti-nutritional factors (ANFs) and toxins, improve the feed flavor, and ultimately enhance the growth performance and intestinal health of pigs.

[0044] The present invention adopts the following technical solutions: In a first aspect, the present invention provides a complete feed fermented by composite bacteria and enzymes and a preparation method thereof.

[0045] (1) The composite bacterial enzyme synergistic fermentation agent (bacterial enzyme composite microecological preparation) is composed of a composite bacterial agent and a composite enzyme preparation. Compound microbial agent: Before mixing, the viable bacterial counts of the bacterial solutions were as follows: Lactobacillus plantarum with the accession number BNCC336421 ( Lactobacillus plantarum , 4.0×10 9 ~4.5×10 9 CFU / mL), Bacillus subtilis with the deposit number of BNCC109047 ( Bacillus subtilis , 1.0×10 8 ~1.5×10 8 CFU / mL), Saccharomyces cerevisiae with the accession number CICC1355 ( Saccharomyces cerevisiae , 4.0×10 7 ~4.5×10 7 CFU / mL) and Aspergillus niger with the accession number CICC40273 ( Aspergillus niger , 1×10 6 CFU / mL~2×10 6 CFU / mL).

[0046] Complex enzyme preparation: α-amylase (EC 3.2.1.1, 3000U / g), lipase (EC 3.1.1.3, 10000U / g), protease (EC 3.4.22.2, 60000U / g), cellulase (EC 3.2.1.4, 10000U / g), pectinase (EC3.2.1.15, 30000U / g), xylanase (EC 3.2.1.8, 1000000U / g), α-galactosidase (EC 3.2.1.22, 500U / g), β-glucanase (EC 3.2.1.73, 50000U / g) and glucose oxidase (EC 1.1.3.4, 10000U / g).

[0047] (2) Preparation of starter culture: The activated Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus niger liquid are uniformly mixed in a volume ratio of 1:2:3:3 to obtain a composite bacterial liquid, and a composite enzyme preparation is added to obtain a composite bacterial enzyme synergistic fermentation agent.

[0048] The composite bacterial solution and the composite enzyme preparation were mixed in a ratio of 9 mL: 3.49 g.

[0049] In a second aspect, the present invention provides a solid bacterial enzyme fermented complete feed for growing pigs, which is a solid feed obtained by fermenting the complete feed with the composite bacterial enzyme solution as described above.

[0050] In a third aspect, the present invention provides a method for preparing a complete feed by synergistic fermentation with solid-state bacterial enzymes.

[0051] The feed is prepared by mixing and fermenting pig feed (preferably corn-soybean meal type feed) and the composite bacteria-enzyme synergistic fermentation agent described in the first aspect.

[0052] Furthermore, the method for preparing liquid fermented feed for growing pigs comprises the following steps: (1) Preparing the composite bacterial enzyme solution for solid-state fermentation feed; (2) Inoculating 9.3% (mL / g) of the compound bacterial enzyme solution prepared in step (1) into the complete feed (pig feed); (3) The feed is mixed with water to a final moisture content of 72%; (4) After mixing evenly, seal the bag and ferment the feed at 36°C for 4.1 days.

[0053] Preferably, the fermentation substrate: based on the total mass of the components of the fermentation substrate as 100%, the fermentation substrate includes the following components: 67.46% corn, 3% whey powder, 1.23% soybean oil, 20.61% soybean meal, 2% fish meal, 1.57% calcium hydrogen phosphate, 0.48% stone powder, 0.56% sodium chloride, 0.55% lysine phosphate, 0.23% methionine, 0.23% threonine, 0.08% tryptophan and 2% premix.

[0054] Preferably, in step (1), the composite bacterial enzyme solution for solid-state fermentation feed as described above is prepared as follows: Lactobacillus plantarum is inoculated into MRS culture medium and cultured statically in a constant temperature box; Bacillus subtilis is inoculated into NB culture medium and cultured on a shaking table; Saccharomyces cerevisiae is inoculated into MEB culture medium and cultured on a shaking table; Aspergillus niger is inoculated into PDB liquid culture medium and cultured on a shaking table. The volume ratio of the obtained activated bacterial solution of Lactobacillus plantarum, activated bacterial solution of Bacillus subtilis, activated bacterial solution of Saccharomyces cerevisiae and activated bacterial solution of Aspergillus niger is 1:2:3:3.

[0055] Thirdly, the solid-state bacterial enzyme-cooperated fermentation complete feed for pigs prepared by the present invention improves the nutritional value of the feed.

[0056] By utilizing the above-mentioned fermentation agent, the present invention has developed a solid-state fermented complete feed for pigs with significantly improved nutritional value and feeding effect.

[0057] (1) The present invention significantly increases the content of TCA-SP, CP, EE and Ash in feed; (2) The present invention significantly reduces the contents of moisture, NDF (neutral detergent fiber) and CF (crude fiber), while significantly degrading various ANFs and toxins DON and ZEN; (3) The macromolecular proteins in the feed of the present invention are degraded into small peptide molecules that are easily absorbed; (4) The present invention improves microbial diversity and screens out 17 differential biomarkers such as Lactobacillus and Pediococcus; (5) The present invention screened out 116 significantly different flavor substances, including dimethyl trisulfide, and the changes in 2-octenal and vanillin made the feed more sweet, fruity, grassy and waxy, while ethyl acetyl octanoate, 3-nonanone and phenylacetaldehyde were the main contributors to the unique fermentation flavor of the feed.

[0058] In a fourth aspect, the present invention provides the use of the above-mentioned solid-state bacterial enzyme-co-fermented complete feed for pigs in pig farming.

[0059] This fermented feed, which replaces 10-15% of complete feed, can significantly improve the growth performance of growing pigs, enhance the body's antioxidant capacity, and effectively improve small intestinal tissue morphology (such as the villus height / crypt depth ratio) and intestinal barrier function.

[0060] In a specific embodiment of the present invention, the liquid fermented complete feed is applied to growing pig farming. Preferably, solid fermented complete feed is used to replace 10% of the complete feed for feeding.

[0061] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0062] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0063] In the present invention, Lactobacillus plantarum with the preservation number of BNCC336421 and Bacillus subtilis with the preservation number of BNCC109047 were purchased from BNCC Culture Collection Center; Saccharomyces cerevisiae with the preservation number of CICC1355 and Aspergillus niger with the preservation number of CICC40273 were purchased from CICC Culture Collection Center.

[0064] Example 1 Establishment and optimization of solid-state bacterial enzyme fermentation process Reagents such as MRS medium, beef extract, peptone, agar, and yeast powder were purchased from Solebao. Growing pig feed was purchased from Jinzhong Shiyang Feed Co., Ltd. and mainly consisted of the following components: 67.46% corn, 3% whey powder, 1.23% soybean oil, 20.61% soybean meal, 2% fish meal, 1.57% calcium hydrogen phosphate, 0.48% stone powder, 0.56% sodium chloride, 0.55% lysine phosphate, 0.23% methionine, 0.23% threonine, 0.08% tryptophan, and 2% premix.

[0065] Among them, the premix provides vitamin A 10,000 IU, vitamin D 3,500 IU, vitamin E 70 IU, vitamin K3 3 mg, vitamin B1 3 mg, vitamin B2 10 mg, vitamin B6 6 mg, biotin 0.4 mg, pantothenic acid 30 mg, niacin 30 mg, copper (copper sulfate pentahydrate) 110 mg, manganese (manganese sulfate) 10 mg, iron (ferrous sulfate) 140 mg, iodine (potassium iodide) 0.5 mg, and selenium (sodium selenite) 0.3 mg per kilogram of feed.

[0066] (1) Bacteria activation a. Activation of Lactobacillus plantarum: Dissolve lyophilized Lactobacillus plantarum (BNCC336421) in 0.5 mL of MRS liquid medium (10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 0.2 g magnesium sulfate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 2.0 g potassium dihydrogen phosphate, 0.04 g manganese sulfate, 0.1 g Tween 80, and 1.0 L distilled water) and mix thoroughly. Then, pipette 0.2 mL of the bacterial suspension onto a solid medium plate (liquid medium plus 20.0 g agar) and spread evenly with a spreader. Incubate the plate in a constant temperature incubator at 37°C for 24 hours. The resulting viable cell count of the Lactobacillus plantarum suspension is approximately 4.3 × 10 9 CFU / mL.

[0067] b. Activation of Bacillus subtilis: Dissolve lyophilized Bacillus subtilis (BNCC135034) powder in 0.5 mL of nutrient agar liquid medium (3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, 1.0 L distilled water) and mix thoroughly. Then, pipette 0.2 mL of the bacterial suspension onto a solid medium plate (liquid medium plus 20.0 g agar) and spread evenly with a spreader. Incubate the plate in a constant temperature incubator at 37°C for 24 hours. The viable count of the resulting Bacillus subtilis suspension is approximately 1.1 × 10 8 CFU / mL.

[0068] c. Activation of Saccharomyces cerevisiae: Dissolve lyophilized Saccharomyces cerevisiae (CICC1355) powder in 0.5 mL of MEB liquid medium (3.0 g beef extract, 10.0 g peptone, 5.0 g lactose, 5.0 g NaCl, 1.0 L distilled water) and mix thoroughly. Then, pipette 0.2 mL of the bacterial suspension onto a solid medium plate (liquid medium plus 20.0 g agar) and spread evenly with a spreader. Incubate the plate in a constant temperature incubator at 37°C for 24 hours. The resulting viable Saccharomyces cerevisiae suspension should contain approximately 4.0 × 10 7 CFU / mL.

[0069] d. Activation of Aspergillus niger: Dissolve lyophilized Aspergillus niger (CICC40273) powder in 0.5 mL of PDB liquid medium (6.0 g / L potato extract, 20 g / L glucose, 1.0 L distilled water) and mix thoroughly. Then, pipette 0.2 mL of the bacterial suspension onto a solid medium plate (liquid medium plus 20.0 g agar) and spread evenly with a spreader. Incubate the plate in a constant temperature incubator at 37°C for 24 hours. The viable count of the resulting Aspergillus niger suspension is approximately 1 × 106 CFU / mL.

[0070] (2) Freezing and resuscitation of bacterial strains: Pick 1-2 colonies from the plate and wash them with an appropriate amount of 20% sterile glycerol solution into 1.5 mL cryovials, seal with sealing film, and freeze at -80°C. Before each fermentation, take one tube of each bacterial strain and thaw it quickly in a water bath within 1-2 minutes. After thawing, add the four resuscitated bacterial solutions to their respective liquid culture media, mix them evenly, and place them in a shaking incubator at 37°C for 24 hours.

[0071] (3) Optimization of fermentation strains The inoculation ratio of the four strains was designed according to an orthogonal experiment as shown in Table 1. The initial inoculum size was 3%, the fermentation temperature was 36°C, the water content was 80%, and the pH value was natural. The feed was inoculated into a 300 mL conical flask containing 50 g of the feed in Example 1. The fermentation was carried out for 72 h using a double-layer sterile sealing film to prepare the fermented feed. The acid-soluble protein, crude fiber and anti-nutritional factor β-conglycinin of the feed were used as the measurement indicators to optimize the strain ratio.

[0072] As shown in Tables 2 and 3, the optimal combination for acid-soluble protein was A1B2C3D3, while the optimal combination for crude fiber degradation was A1B3C3D3. Furthermore, the A1B2C3D3 group exhibited superior glycinin and β-conglycinin degradation to the A1B3C3D3 group. Therefore, the fermentation combination A1B2C3D3 was ultimately determined to be the optimal mixed bacterial ratio for subsequent screening.

[0073] The effects of mixed fermentation with different ratios on anti-nutrient factors are shown in Figure 1 .

[0074] (4) Optimization of fermentation conditions The optimal strain ratios screened above were optimized by single factor and response surface optimization experiments ( Figure 2 and Figure 3 ) The contents of acid-soluble protein and crude fiber in feed were detected under different fermentation times (1 d, 2 d, 3 d, 4 d, 5 d, 6 d), fermentation temperatures (24 ℃, 28 ℃, 32 ℃, 36 ℃, 40 ℃), moisture contents (60%, 80%, 100%, 120%, 140%) and bacterial inoculum sizes (6%, 9%, 12%, 15%, 18%).

[0075] from Figure 2It can be seen from the AD that under different fermentation conditions, the most suitable water content is 80%, the inoculation amount is 9%, the fermentation time is 4 days, and the fermentation temperature is 36 ℃. At the same time, the response surface optimization software predicts that the optimal acid-soluble protein value is 4.112% when the inoculation amount is 9.283%, the water content is 71.539%, and the fermentation time is 4.112 days. Figure 3 , AC).

[0076] In addition, in order to solve the problem of insufficient enzyme production during mixed bacteria fermentation, a complex enzyme preparation containing 9 enzymes was used for bacterial and enzyme synergistic fermentation with mixed bacteria fermentation. Figure 4 It proves that the fermentation effect of bacteria and enzymes is better.

[0077] Complex enzyme preparation: α-amylase (EC 3.2.1.1, 3000U / g), lipase (EC 3.1.1.3, 10000U / g), protease (EC 3.4.22.2, 60000U / g), cellulase (EC 3.2.1.4, 10000U / g), pectinase (EC3.2.1.15, 30000U / g), xylanase (EC 3.2.1.8, 1000000U / g), α-galactosidase (EC 3.2.1.22, 500U / g), β-glucanase (EC 3.2.1.73, 50000U / g) and glucose oxidase (EC 1.1.3.4, 10000U / g).

[0078] The composite bacterial solution and the composite enzyme preparation were mixed at a ratio of 9 mL:3.49 g for bacterial-enzyme synergistic fermentation.

[0079] (5) Preparation method of solid-state fermentation feed for pigs Based on the above optimization results, the preparation method of bacterial enzyme solid fermentation feed is as follows: the expanded Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae and Aspergillus niger liquid are mixed evenly in a volume ratio of 1:2:3:3 to obtain a composite bacterial liquid. Among them, the viable cell count of the Bacillus subtilis liquid is not less than 1.2×10 9 CFU / mL, the viable count of Lactobacillus plantarum liquid was determined to be no less than 3.7×10 9 CFU / mL, the viable count of Saccharomyces cerevisiae is not less than 4.0×10 7 CFU / mL, the viable count of Aspergillus niger was determined to be no less than 1×10 6 CFU / mL.

[0080] The optimal bacterial strain ratio and fermentation conditions for growing pigs are obtained according to orthogonal experiments, single factor and response surface optimization experiments to prepare a mixed bacterial liquid, and the composite bacterial liquid is then inoculated into a mixture of feed and water at an inoculum amount of 10% (V / W, L / kg, ml / g); after uniform mixing, the sealed bag is sealed and the mixture is placed in a constant temperature electric heating incubator for fermentation at 36°C for 4.1 days to obtain a bacterial enzyme solid-state fermentation feed for growing pigs (a bacterial enzyme solid-state fermentation feed for pigs).

[0081] Example 2 Quality of Pig Feed Solid-State Fermented with Fungal Enzymes The fungal enzyme solid-state fermentation feed for growing pigs prepared in Example 1 was compared with a control group (ordinary complete feed, i.e., the pig feed in Example 1) to verify the feed quality.

[0082] 1. Materials Lactobacillus plantarum (BNCC336421) and Bacillus subtilis (BNCC109047) were purchased from BNCC Beina Biotechnology Co., Ltd., while Saccharomyces cerevisiae (CICC1355) and Aspergillus niger (CICC40273) were purchased from the China Industrial Microbiological Culture Collection (CICC). ELISA kits for glycinin, trypsin inhibitor, β-conglycinin, phytohemagglutinin, deoxynivalenol, zearalenone, aflatoxins, and T-2 toxin were purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0083] 2. Methods (1) Nutritional components of bacterial enzyme solid-state fermentation feed As shown in Table 4, the contents of crude protein, crude fat and ash in the solid fermentation feed were significantly higher than those in the control group ( P <0.01), and the contents of water, neutral detergent fiber and acid detergent fiber were significantly lower than those in the control group ( P <0.01). Scanning electron microscopy and gel electrophoresis results showed that the feed was fully lysed after solid-state fermentation and most of the macromolecular proteins were degraded and converted into small molecular proteins that are easily absorbed after solid-state fermentation ( Figure 5 , AB).

[0084] (2) Anti-nutritional factor content of bacterial enzyme solid-state fermentation As can be seen from Tables 5 and 6, all anti-nutritional components in the fermented feed group, including α-conglycinin, β-conglycinin, SPag, TI, and PHA, were significantly reduced ( P <0.01), and the contents of toxins DON and ZEN were significantly reduced compared with the control group (P <0.01).

[0085] The bacterial enzyme solid-state fermentation feed improves the nutritional value of the complete feed and reduces the anti-nutritional factor content of the feed. The loose and porous surface structure of the feed after fermentation increases the opportunity for the enzyme to fully utilize the feed substrate, thereby better improving its nutritional value.

[0086] Example 3 Effect of solid-state fermentation feed with bacterial enzymes on growing pigs (1) Microbial community structure of solid-state fermentation feed from Figure 6 It can be seen that the α diversity of the solid-state fermentation feed group increased significantly, and the microbial community structures between the two groups were clearly separated ( Figure 6 , AD). The Venn diagram showed that the control group had 2,574 ASVs, while the solid-state fermentation group had 4,117 ASVs ( Figure 7 , AB). At the phylum level, the proportions of Cyanobacteria and Proteobacteria in the solid-state fermentation group were significantly reduced ( P <0.01, while the proportions of Firmicutes and Acidobacteria increased significantly ( P <0.01, Figure 8 At the genus level, the core genus of the solid-state fermentation group is Lactobacillus ( P <0.01), and significantly increased the proportion of lactic acid bacteria and Pediococcus, and significantly reduced the proportion of Streptococcus ( Figure 8 LEfSe analysis showed that the control group had 10 specific microbial genera, while the solid-state fermentation group had 17 differentially abundant bacterial communities ( Figure 9 , AB), and are mainly enriched in amino acid and nucleotide sugar pathways ( Figure 10 , AC).

[0087] (2) Feed flavor metabolites from solid-state fermented feed from Figure 11 It can be seen that the volatile compounds in each group of feeds clustered together, while the two groups were clearly separated, indicating that the data are reliable ( Figure 11 , AB). The Venn diagram showed that 404 specific compounds were identified in the control group and 513 specific compounds were identified in the solid-state fermentation group ( Figure 11 C), and the relative contents of aldehydes, acids, esters and phenolic flavor compounds in the feed increased after fermentation ( Figure 11 ROAV analysis showed that the relative contents of 2-octenal and vanillin in the solid-state fermentation group increased significantly ( Figure 12 A), which also enhances sweet, fruity, and fatty flavors ( Figure 12 The volcano plot results showed that the solid-state fermentation group significantly downregulated several flavor compounds, including ethyl dodecanoate and methionine ( Figure 13 ), while more than five flavor metabolites were associated with sensory characteristics including ethyl acetyloctanoate, 3-nonanone, phenylacetaldehyde, and acetic acid ( Figure 14 ).

[0088] Example 4 Effect of solid-state fermentation feed with bacterial enzymes on growing pigs To verify the efficacy of the solid-state fermented feed prepared in Example 1 on growing pigs, 32 healthy Duroc x Jinfen White pigs, aged 60 ± 3 days and weighing 17.87 ± 2.83 kg, were randomly divided into two treatment groups, each with four replicates. The control group was fed a complete feed, while the experimental group was fed a feed consisting of 10% of the solid-state fermented feed prepared in Example 1 mixed with 90% of the basal diet.

[0089] Each group of pigs was divided into four pens, each housed individually. All pigs had free access to water and food. Feed was administered daily in the morning and evening, and the remaining feed in the trough was monitored. The remaining feed was weighed and recorded before the next feeding, which was used to determine the next feeding amount. The temperature, humidity, and odor of the pigpen were monitored daily, and ventilation was maintained. The pre-feeding period lasted five days, and the actual trial lasted 28 days. The pigs were then slaughtered at the end of the trial. The feeding results are as follows: (1) Growth performance of growing pigs As can be seen from Table 7, compared with the control group, the final weight and ADG of growing pigs fed SSF diet were significantly increased ( P <0.05), FCR was significantly reduced ( P <0.01), and ADFI had no significant effect.

[0090] (2) Serum biochemistry of growing pigs As can be seen from Table 8, the levels of albumin and blood glucose in the serum of the solid-state fermentation group were significantly increased compared with the control group ( P <0.01), globulin and creatinine decreased significantly ( P There was no significant effect on total protein, glutamyl transferase, alanine aminotransferase, alanine aminotransferase, urea, insulin, and growth hormone.

[0091] As can be seen from Table 9, compared with the control group, the serum IgA of growing pigs in the SSF group ( P <0.01) and IgG ( P <0.05) significantly increased, and the levels of inflammatory factors IL-4 and IL-6 also decreased significantly ( P<0.01). In addition, the serum antioxidant indexes of SOD and GSH-PX in the solid-state fermentation group were significantly increased ( P <0.05, while the MDA content was significantly decreased ( P <0.05).

[0092] (3) Meat quality of growing pigs As shown in Table 10, the shear force of the longissimus dorsi muscle of the growing pigs in the solid-state fermentation group was significantly lower than that in the control group ( P <0.01), drip loss of meat, pH 45min , pH 24h , and flesh-colored L 1h 、L 24h 、a 1h 、a 24h 、b 1h 、b 24h There was no significant difference.

[0093] (4) Small intestinal barrier function of growing pigs As can be seen from Table 11, the villus height and crypt depth of each intestinal segment of solid-state fermentation were significantly higher than those of the control group ( P <0.01, and the villus-crypt ratio of the jejunum and duodenum was also significantly higher than that of the control group ( P <0.01).

[0094] from Figure 15 (A and B) It can be seen that the villi of the small intestine of the solid-state fermentation group are arranged more neatly, and the villi of the jejunum have more fan-shaped morphology, with a larger absorption area. At the same time, the expression levels of tight junction proteins Claudin-1, Occludin and ZO-1 in the jejunum tissue of the solid-state fermentation group are significantly higher than those of the control group ( P <0.05).

[0095] Example 5 Optimization of fermentation strains In the early stage of the experiment, eight microorganisms were selected for single fermentation, including Pediococcus acidilactici, Bacillus coagulans, Aspergillus niger, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis. The results are shown in Table 12: As can be seen from Table 12, the single fermentation of the eight microorganisms increased the contents of acid-soluble protein, reducing sugar and lactic acid in rapeseed meal ( P<0.05), and reduced the content of β-conglycinin (P<0.05). Compared with the other groups of strains, Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger significantly increased the content of acid-soluble protein and reducing sugar after fermentation ( P <0.05), degraded β-conglycinin content ( P <0.05), while Aspergillus niger, Lactobacillus plantarum and Bacillus subtilis also significantly increased the content of reducing sugars ( P <0.01). Finally, Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger were selected to prepare a composite bacterial agent for bacterial enzyme synergistic fermentation of feed.

[0096] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A bacterial enzyme compound microecological preparation, characterized in that: It is made by mixing compound bacterial agent and compound enzyme preparation in proportion; The composite bacterial agent comprises Bacillus subtilis ( Bacillus subtilis )、Lactobacillus plantarum( Lactobacillus plantarum ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Aspergillus niger ( Aspergillus niger ); The complex enzyme preparation comprises alpha-amylase, lipase, protease, cellulase, pectinase, xylanase, alpha-galactosidase, beta-glucanase and glucose oxidase.

2. The bacterial enzyme composite microecological preparation according to claim 1, characterized in that: The composite bacterial agent and the composite enzyme preparation are mixed in a ratio of 9 mL: 3.49 g; The composite bacterial agent is prepared by mixing Bacillus subtilis liquid, Lactobacillus plantarum liquid, Saccharomyces cerevisiae liquid and Aspergillus niger liquid in a volume ratio of 1:2:3:3; wherein the bacterial content of the Lactobacillus plantarum liquid is 4.0×10 9 ~4.5×10 9 CFU / mL, the bacterial content of Bacillus subtilis liquid was 1.0×10 8 ~1.5×10 8 CFU / mL, the bacterial content of the Saccharomyces cerevisiae liquid was 4.0×10 7 ~4.5×10 7 CFU / mL, the bacterial content of Aspergillus niger liquid is 1×10 6 CFU / mL~2×10 6 CFU / mL; The contents of various enzymes in the complex enzyme preparation are 3000U / g of α-amylase, 10000U / g of lipase, 60000U / g of protease, 10000U / g of cellulase, 30000U / g of pectinase, 1000000U / g of xylanase, 500U / g of α-galactosidase, 50000U / g of β-glucanase and 10000U / g of glucose oxidase.

3. The bacterial enzyme composite microecological preparation according to claim 1 or 2, characterized in that: The accession number of the Lactobacillus plantarum is BNCC336421, the accession number of the Bacillus subtilis is BNCC109047, the accession number of the Saccharomyces cerevisiae is CICC1355, and the accession number of the Aspergillus niger is CICC40273; The EC numbers of the enzymes in the complex enzyme preparation are: α-amylase EC 3.2.1.1, lipase EC 3.1.1.3, protease EC 3.4.22.2, cellulase EC 3.2.1.4, pectinase EC 3.2.1.15, xylanase EC 3.2.1.8, α-galactosidase EC 3.2.1.22, β-glucanase EC 3.2.1.73, and glucose oxidase EC 1.1.3.

4.

4. Application of the bacterial enzyme composite microecological preparation according to any one of claims 1 to 3 in the field of feed processing.

5. Solid-state bacterial enzyme synergistic fermentation feed, characterized in that: It is prepared by mixing and fermenting the bacterial enzyme composite microecological preparation according to any one of claims 1 to 3 with feed; Preferably, the fermentation method is solid-state fermentation; Preferably, the feed is a corn-soybean meal type feed.

6. The feed according to claim 5, characterized in that The amount of the bacterial enzyme compound probiotic preparation added to the corn-soybean meal feed is 9 mL; Preferably, the corn-soybean meal feed includes the following components in grams: 67.46g corn, 3g whey powder, 1.23g soybean oil, 20.61g soybean meal, 2g fish meal, 1.57g calcium hydrogen phosphate, 0.48g stone powder, 0.56g sodium chloride, 0.55g lysine phosphate, 0.23g methionine, 0.23g threonine, 0.08g tryptophan and 2g premix.

7. The method for preparing the feed according to claim 5 or 6, characterized in that: The bacterial enzyme composite microecological preparation and the corn-soybean meal feed were mixed evenly in proportion, water was added to adjust the moisture content to 72%, and the mixture was placed under sealed conditions at 36° C. and fermented for 4.1 days.

8. Any of the following applications of the preparation method according to claim 7: 1) Used to increase the content of ash, acid-soluble protein, OM, CP and EE in corn-soybean meal feed, thereby improving the nutritional value of the feed; 2) Used to reduce the content of anti-nutritional factors and toxins in corn-soybean meal feed; 3) Used to increase the abundance of beneficial bacteria in corn-soybean meal feed; 4) Used to increase the content of aldehyde, acid, ester and phenolic flavor compounds in fermented corn-soybean meal feed, making the feed have sweet, fruity, grassy and waxy flavors, and improving the palatability of the feed; Preferably, the anti-nutritional factors in 2) include α-conglycinin, β-conglycinin, soybean antigen protein, trypsin inhibitor TI and lectin, and the toxins include DON and ZEN; Preferably, the beneficial bacteria in 3) include Lactobacillus ( Lactobacillus ) and Pediococcus ( Pediococcus )microorganism.

9. Any of the following uses of the feed according to claim 5 or 6: (1) Used in pig farming; (2) Used to increase the average daily weight gain of young pigs and reduce feed conversion rate; (3) Used to increase the villus height, crypt depth and villus-to-crypt ratio of the small intestine of growing pigs; (4) Used to increase the expression of pig intestinal tight junction proteins Claudin-1, Occludin and ZO-1.

10. The use according to claim 9, characterized in that (1) The feed is used to replace 10-15% of the complete feed.

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