Bacillus licheniformis fermented mixed feed and preparation method thereof

Abstract

The invention relates to the technical field of animal feed, in particular to bacillus licheniformis fermented mixed feed and a preparation method thereof. The method comprises the following steps: mixing a plurality of different microbial strains to prepare a fermentation mixed seed solution; then, chitosan is subjected to sulfhydrylation modification through L-cysteine, and modified chitosan is obtained; then taking the modified chitosan and zein as wall materials and taking the compound enzyme as a core material to obtain the immobilized compound enzyme, so that efficient immobilization and protection of the compound enzyme are realized; finally, the fermentation base material is fully fermented under the synergistic effect of bacteria and enzymes, so that the fermented mixed feed is obtained, and the nutritional value and the utilization rate of the feed are favorably improved.

Description

Technical Field

[0001] The invention relates to the technical field of animal feed, in particular to a Bacillus licheniformis fermented mixed feed and a preparation method thereof. Background Art

[0002] Bacillus licheniformis is a common probiotic with multiple physiological activities and nutritional functions. Its fermentation products can be used as feed additives to improve the digestion and absorption capacity of animals, enhance immunity and promote growth and development. Bacillus licheniformis fermented mixed feed is a nutritious feed obtained by mixing Bacillus licheniformis with other feed ingredients for fermentation.

[0003] With the development of animal husbandry and the increasing demand for animal nutrition, Bacillus licheniformis fermented mixed feed has become a new type of feed with broad market prospects. It can improve animal production performance, reduce feed waste, and improve meat quality. However, some Bacillus licheniformis fermented mixed feeds have unreasonable ratios of other ingredients, resulting in unbalanced nutrients, which affects the animal's dietary intake and growth and development. In addition, the enzymes added to the feed are easily affected by factors such as temperature, pH value and antibiotics, resulting in reduced activity and stability, thereby affecting the fermentation efficiency.

[0004] Therefore, we propose a Bacillus licheniformis fermented mixed feed and a preparation method thereof. Summary of the invention

[0005] The object of the present invention is to provide a Bacillus licheniformis fermented mixed feed and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for preparing a mixed feed fermented by Bacillus licheniformis comprises the following steps:

[0008] Step S1: activating Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, and yeast and inoculating them into seed culture medium for seed culture to obtain Bacillus subtilis seed liquid, Bacillus licheniformis seed liquid, lactic acid bacteria seed liquid, and yeast seed liquid; mixing the Bacillus subtilis seed liquid, Bacillus licheniformis seed liquid, lactic acid bacteria seed liquid, and yeast seed liquid uniformly to obtain a fermentation mixed seed liquid;

[0009] Step S2: Evenly mix the fermentation mixed seed liquid and the fermentation base material to obtain a mixed fermentation base material; then add the immobilized complex enzyme and mix evenly, adjust the water content of the mixed fermentation base material to 40-50%, and ferment at 20-30° C. for 5-7 days to obtain a fermented mixed feed.

[0010] Furthermore, the specific steps of activation in step S1 are: inoculating and streaking Bacillus subtilis and Bacillus licheniformis into beef extract peptone medium respectively, and culturing at 30-37°C for 1-2 days; inoculating and streaking lactic acid bacteria into MRS medium, and culturing anaerobically at 30-35°C for 3-5 days; inoculating and streaking yeast into PDA medium, and culturing at 28-30°C for 1-2 days.

[0011] Furthermore, the beef extract peptone culture medium is: beef extract 3-5g / L, peptone 10-15g / L, sodium chloride 3-5g / L, agar 15-20g / L, pH=7.2-7.4, sterilized at 121°C for 15-20min.

[0012] Further, the MRS medium is: peptone 5-10g / L, beef extract 10-12g / L, yeast extract 2.5-5.0g / L, diammonium citrate 2-4g / L, glucose 20-22g / L, Tween 80 1-2g / L, sodium acetate trihydrate 4-6g / L, dipotassium hydrogen phosphate trihydrate 2-4g / L, magnesium sulfate heptahydrate 0.55-0.58g / L, manganese sulfate monohydrate 0.25-0.28g / L, agar 15-20g / L, pH=6.2-6.5, sterilized at 121°C for 15-20min.

[0013] Furthermore, the PDA culture medium is: 200-202 g / L of peeled potatoes, 18-20 g / L of glucose, and 15-20 g / L of agar, sterilized at 121° C. for 15-20 min.

[0014] Furthermore, the specific steps of seed culture in step S1 are: inoculating and streaking the activated Bacillus subtilis and Bacillus licheniformis into the seed culture medium respectively, and shaking and culturing them at 30-37°C and 160-180r / min for 1-2d to obtain Bacillus subtilis seed solution and Bacillus licheniformis seed solution; inoculating and streaking the activated lactic acid bacteria into the seed culture medium, and shaking and culturing them at 30-37°C and 160-180r / min for 2-3d to form lactic acid bacteria seed solution; inoculating and streaking the activated yeast into the seed culture medium, and shaking and culturing them at 28-30°C and 160-180r / min for 2-3d to form yeast seed solution.

[0015] Furthermore, the seed culture medium of Bacillus subtilis and Bacillus licheniformis is: 3-5 g / L beef extract, 10-15 g / L peptone, 3-5 g / L sodium chloride, pH=7.2-7.4; the seed culture medium of lactic acid bacteria is: 5-10 g / L peptone, 10-12 g / L beef extract, 2.5-5.0 g / L yeast extract, 2-4 g / L diammonium hydrogen citrate, 20-22 g / L glucose, 1-2 g / L Tween 80, 4-6 g / L sodium acetate trihydrate, 2-4 g / L dipotassium hydrogen phosphate trihydrate, 0.55-0.58 g / L magnesium sulfate heptahydrate, 0.25-0.28 g / L manganese sulfate monohydrate, pH=6.2-6.5; the seed culture medium of yeast is: 200-202 g / L peeled potato, 18-20 g / L glucose.

[0016] Furthermore, the mass ratio of each bacterial species in the fermentation mixed seed liquid in step S1 is: Bacillus subtilis: Bacillus licheniformis, lactic acid bacteria, yeast = 1: (1-2): (1.0-1.5): (0.5-0.8).

[0017] Furthermore, the total number of colonies in the fermented mixed seed solution in step S1 is 1×10 6 -1×10 8 CFU / g.

[0018] Furthermore, the preparation method of the immobilized complex enzyme in step S2 is as follows:

[0019] Step (1): mixing chitosan and hydrochloric acid solution uniformly, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and then adding L-cysteine, adjusting the pH value to 5-6, reacting for 3-5 hours, dialyzing, and freeze-drying to obtain modified chitosan;

[0020] Step (2): uniformly mix the modified chitosan and deionized water, adjust the pH value of the system to 4.0-4.4, add the mixed solution of the complex enzyme solution, zein and ethanol under the condition of high-speed shearing at 3000-4000 r / min, stir for 3-5 hours, and obtain the immobilized complex enzyme after rotary evaporation, centrifugation and drying.

[0021] In the above technical scheme, chitosan is modified by thiolization with L-cysteine ​​to obtain modified chitosan with better biocompatibility, stability and controllability; then, nanoparticles loaded with complex enzymes are prepared by the anti-solvent method using modified chitosan and zein as wall materials and complex enzymes as core materials, i.e., immobilized complex enzymes, thereby achieving efficient fixation and protection of the complex enzymes and facilitating improvement of the enzyme loading capacity and stability.

[0022] Furthermore, in step (1), the mass ratio of chitosan to hydrochloric acid solution is 1:(8-10), and the concentration of the hydrochloric acid solution is 0.09-0.10 mol / L.

[0023] Furthermore, in step (1), the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 2.0-2.5 times the mass of chitosan.

[0024] Furthermore, in step (1), the mass of N-hydroxysuccinimide is 0.8-1.0 times the mass of chitosan.

[0025] Furthermore, in step (1), the mass of L-cysteine ​​is 1.0-1.2 times the mass of chitosan.

[0026] Furthermore, in the step (2), the mass ratio of the modified chitosan to deionized water is 1:(250-300).

[0027] Furthermore, in step (2), the composite enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase and xylanase in an enzyme activity ratio of 1:(1-2):1:1:(0.5-0.6):(8-10).

[0028] Furthermore, the concentration of the complex enzyme solution in step (2) is 1.0-1.2 mg / mL, and its amount is 1-2% of the mass of the modified chitosan.

[0029] Furthermore, in step (2), the mass of zein is 1.0-1.2 times the mass of modified chitosan, and the mass of ethanol is 100-150 times the mass of zein.

[0030] Furthermore, the mass ratio of the fermentation mixed seed liquid to the fermentation base material is 1:(15-25).

[0031] Furthermore, the fermentation base includes the following components by weight: 40-50 parts of corn, 20-30 parts of soybean meal, 10-20 parts of fermented sweet potato residue, 1.0-1.5 parts of calcium hydrogen phosphate, 0.8-1.0 parts of zeolite powder, and 0.3-0.5 parts of salt.

[0032] Furthermore, the fermented sweet potato residue is prepared by mixing sweet potato residue and fermented mixed seed liquid in a mass ratio of 1:(0.04-0.05), adding 500-1000U / g immobilized complex enzyme, and solid fermenting in a breathing bag for 60-65h.

[0033] In the above technical scheme, fermented sweet potato residue is obtained through the synergistic effect of fermented mixed seed liquid and immobilized complex enzyme; using fermented sweet potato residue to replace part of corn and other raw materials can reduce feed costs, improve the economy of feed, and realize efficient resource utilization of sweet potato residue.

[0034] Furthermore, in step S2, the amount of immobilized complex enzyme added is 1000-4000 U / g.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. A Bacillus licheniformis fermented mixed feed and a preparation method thereof. The mixed seed liquid with multiple biological activities can be formed by uniformly mixing Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria and yeast seed liquid, which is beneficial to promoting fermentation and improving the nutritional value of the feed; then, chitosan is thiol-modified by L-cysteine ​​to obtain modified chitosan, so that it has better biocompatibility, stability and controllability and is suitable for being used as a carrier of nanomaterials; then, the modified chitosan and zein are used as wall materials and the complex enzyme is used as the core material, and nanoparticles loaded with the complex enzyme, i.e., immobilized complex enzyme, are prepared by an antisolvent method, so that efficient fixation and protection of the complex enzyme are achieved, which is beneficial to improving the enzyme loading and stability; finally, the fermentation base material is fully fermented by utilizing the synergistic effect of bacteria and enzymes, so as to obtain the fermented mixed feed, which is beneficial to improving the nutritional value and utilization rate of the feed.

[0037] 2. A Bacillus licheniformis fermented mixed feed and a preparation method thereof of the present invention can promote the fermentation process and improve the product quality by adding fermented sweet potato residue; using fermented sweet potato residue to replace part of the corn raw material can effectively reduce the cost of feed and improve the nutritional value of feed, thereby improving the growth performance and health of animals, and further improving the economic benefits of the breeding industry. At the same time, by synergizing the fermented mixed seed liquid with the immobilized complex enzyme to obtain the fermented sweet potato residue, the efficient utilization of the sweet potato residue is achieved, the waste of agricultural by-products is reduced, and it is helpful to achieve the sustainable utilization of resources and the protection of the environment. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In this embodiment, Bacillus subtilis: the deposit number is CGMCC1.821, which is from the China General Microbiological Culture Collection Administration Center; Bacillus licheniformis: the deposit number is CGMCC1.9048, which is from the China General Microbiological Culture Collection Administration Center; lactic acid bacteria: Lactobacillus plantarum, model JYLP-326, which is from Shandong Dawei Pharmaceutical Co., Ltd.; yeast: Saccharomyces cerevisiae (Saccharomyces cerevisiae) cerevisiae), model JYY-01, sourced from Jinan Jinyuyuan Biotechnology Co., Ltd.; corn: corn cob powder, 30-100 mesh, item number 0342, sourced from Jinan Jinyi Chemical Co., Ltd.; soybean meal: item number 7-964, sourced from Jinan Fuqiao Chemical Industry and Trade Co., Ltd.; sweet potato residue: item number 0085, sourced from Qingdao Yutaifeng Trading Co., Ltd.; zeolite powder: item number 561, 800 mesh, sourced from Lingshou County Zenan Mineral Products Processing Co., Ltd.; salt: 100 mesh, model JZY-016, sourced from Laizhou Haimeng Salt Chemical Co., Ltd.

[0040] In the following examples and comparative examples, 1 part is equal to 10 g.

[0041] Example 1: A method for preparing a mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0042] Step S1: inoculate and streak Bacillus subtilis and Bacillus licheniformis into beef extract peptone medium respectively, and culture at 30°C for 1 day; inoculate and streak lactic acid bacteria into MRS medium, and culture at 30°C for 3 days; inoculate and streak yeast into PDA medium, and culture at 28°C for 1 day; inoculate and streak activated Bacillus subtilis and Bacillus licheniformis into seed medium respectively, and culture at 30°C and 160r / min for 1 day to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid; inoculate and streak activated lactic acid bacteria into seed medium, and culture at 30°C and 160r / min for 2 days to form lactic acid bacteria seed liquid; inoculate and streak activated yeast into seed medium, and culture at 28°C and 160r / min for 2 days to form yeast seed liquid;

[0043] Beef extract peptone medium is: beef extract 3g / L, peptone 10g / L, sodium chloride 3g / L, agar 15g / L, pH = 7.2, sterilized at 121℃ for 15min; MRS medium is: peptone 5g / L, beef extract 10g / L, yeast extract 2.5g / L, diammonium hydrogen citrate 2g / L, glucose 20g / L, Tween 80 1g / L, sodium acetate trihydrate 4g / L, dipotassium hydrogen phosphate trihydrate 2g / L, magnesium sulfate heptahydrate 0.55g / L, manganese sulfate monohydrate 0.25g / L, agar 15g / L, pH = 6.2, sterilized at 121℃ for 15min; PDA medium is: peeled potato 200g / L, glucose 18g / L, agar 15g / L, sterilized at 121℃ for 15min;

[0044] The seed culture medium of Bacillus subtilis and Bacillus licheniformis is: 3g / L beef extract, 10g / L peptone, 3g / L sodium chloride, pH=7.2; the seed culture medium of lactic acid bacteria is: 5g / L peptone, 10g / L beef extract, 2.5g / L yeast extract, 2g / L diammonium hydrogen citrate, 20g / L glucose, 1g / L Tween 80, 4g / L sodium acetate trihydrate, 2g / L dipotassium hydrogen phosphate trihydrate, 0.55g / L magnesium sulfate heptahydrate, 0.25g / L manganese sulfate monohydrate, pH=6.2; the seed culture medium of yeast is: 200g / L peeled potato, 18g / L glucose;

[0045] The Bacillus subtilis seed solution, the Bacillus licheniformis seed solution, the lactic acid bacteria seed solution and the yeast seed solution are uniformly mixed to obtain a fermentation mixed seed solution (the mass ratio of each strain is: Bacillus subtilis: Bacillus licheniformis, lactic acid bacteria, yeast = 1:1:1:0.5);

[0046] Step S2: mixing the fermentation mixed seed liquid and the fermentation base material (the fermentation base material includes the following weight components: 40 parts of corn, 20 parts of soybean meal, 10 parts of fermented sweet potato residue, 1.0 part of calcium hydrogen phosphate, 0.8 parts of zeolite powder, and 0.3 parts of salt) at a mass ratio of 1:15 to obtain a mixed fermentation base material; then adding 1000U / g of immobilized complex enzyme to mix evenly, adjusting the water content of the mixed fermentation base material to 40%, and fermenting at 20°C for 5 days to obtain a fermented mixed feed;

[0047] The fermented sweet potato residue was prepared by mixing 10 parts of sweet potato residue and 0.4 parts of fermentation mixed seed liquid, adding 500U / g immobilized complex enzyme, and solid fermenting in a breathing bag for 60h.

[0048] The preparation method of the immobilized complex enzyme in step S2 is as follows:

[0049] Step (1): uniformly mix 1 part of chitosan and 8 parts of 0.09 mol / L hydrochloric acid solution, add 2 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.8 parts of N-hydroxysuccinimide, and then add 1 part of L-cysteine, adjust the pH value to 5, react for 3 hours, dialyze, and freeze-dry to obtain modified chitosan;

[0050] Step (2): 1 part of modified chitosan and 250 parts of deionized water are mixed evenly, the pH value of the system is adjusted to 4.0, and under the condition of 3000r / min high-speed shearing, 0.01 parts of 1mg / mL complex enzyme solution (the complex enzyme is compounded by amylase, cellulase, β-mannanase, acid protease, pectinase, and xylanase according to the enzyme activity ratio of 1:1:1:1:0.5:8), 1 part of zein and 100 parts of ethanol are added, and the mixture is stirred for 3h. After rotary evaporation, centrifugation and drying, an immobilized complex enzyme is obtained.

[0051] Example 2: A method for preparing a mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0052] Step S1: inoculate and streak Bacillus subtilis and Bacillus licheniformis into beef extract peptone medium respectively, and culture at 35°C for 1.5 days; inoculate and streak lactic acid bacteria into MRS medium, and culture at 32°C for 4 days; inoculate and streak yeast into PDA medium, and culture at 29°C for 1.5 days; inoculate and streak activated Bacillus subtilis and Bacillus licheniformis into seed medium respectively, and culture at 35°C and 170r / min for 1.5 days to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid; inoculate and streak activated lactic acid bacteria into seed medium, and culture at 35°C and 170r / min for 2.5 days to form lactic acid bacteria seed liquid; inoculate and streak activated yeast into seed medium, and culture at 29°C and 170r / min for 2.5 days to form yeast seed liquid;

[0053] Beef extract peptone medium is: beef extract 4g / L, peptone 12g / L, sodium chloride 4g / L, agar 18g / L, pH=7.3, sterilized at 121℃ for 18min; MRS medium is: peptone 8g / L, beef extract 11g / L, yeast extract 4g / L, diammonium hydrogen citrate 3g / L, glucose 21g / L, Tween 80 1.5g / L, sodium acetate trihydrate 5g / L, dipotassium hydrogen phosphate trihydrate 3g / L, magnesium sulfate heptahydrate 0.56g / L, manganese sulfate monohydrate 0.27g / L, agar 16g / L, pH=6.4, sterilized at 121℃ for 18min; PDA medium is: peeled potato 201g / L, glucose 19g / L, agar 18g / L, sterilized at 121℃ for 18min;

[0054] The seed culture medium of Bacillus subtilis and Bacillus licheniformis is: beef extract 4g / L, peptone 12g / L, sodium chloride 4g / L, pH=7.3; the seed culture medium of lactic acid bacteria is: peptone 8g / L, beef extract 11g / L, yeast extract 4g / L, diammonium hydrogen citrate 3g / L, glucose 21g / L, Tween 80 1.5g / L, sodium acetate trihydrate 5g / L, dipotassium hydrogen phosphate trihydrate 3g / L, magnesium sulfate heptahydrate 0.57g / L, manganese sulfate monohydrate 0.27g / L, pH=6.4; the seed culture medium of yeast is: peeled potato 202g / L, glucose 20g / L;

[0055] The Bacillus subtilis seed solution, the Bacillus licheniformis seed solution, the lactic acid bacteria seed solution and the yeast seed solution were mixed evenly to obtain a fermentation mixed seed solution (the mass ratio of each strain was: Bacillus subtilis: Bacillus licheniformis, lactic acid bacteria, yeast = 1:1.5:1.2:0.6);

[0056] Step S2: mixing the fermentation mixed seed liquid and the fermentation base material (the fermentation base material includes the following weight components: 45 parts of corn, 25 parts of soybean meal, 15 parts of fermented sweet potato residue, 1.2 parts of calcium hydrogen phosphate, 0.9 parts of zeolite powder, and 0.4 parts of salt) at a mass ratio of 1:20 to obtain a mixed fermentation base material; then adding 2500U / g of immobilized complex enzyme and mixing evenly, adjusting the water content of the mixed fermentation base material to 45%, and fermenting at 25°C for 6 days to obtain a fermented mixed feed;

[0057] The fermented sweet potato residue was prepared by mixing 15 parts of sweet potato residue and 0.7 parts of fermented mixed seed liquid, adding 800U / g of immobilized complex enzyme, and solid fermenting in a breathing bag for 63h.

[0058] The preparation method of the immobilized complex enzyme in step S2 is as follows:

[0059] Step (1): 2 parts of chitosan and 18 parts of 0.1 mol / L hydrochloric acid solution are mixed uniformly, 4.8 parts of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1.8 parts of N-hydroxysuccinimide, and 2.2 parts of L-cysteine ​​are added, the pH value is adjusted to 5-6, the reaction is carried out for 3-5 hours, and after dialysis and freeze-drying, modified chitosan is obtained;

[0060] Step (2): 2 parts of modified chitosan and 550 parts of deionized water are mixed evenly, the pH value of the system is adjusted to 4.2, and under the condition of 3500r / min high-speed shearing, 0.06 parts of 1.1mg / mL complex enzyme solution (the complex enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase, and xylanase according to the enzyme activity ratio of 1:1.5:1:1:0.55:9), 2.2 parts of zein and 300 parts of ethanol are added, and the mixture is stirred for 4h. After rotary evaporation, centrifugation and drying, an immobilized complex enzyme is obtained.

[0061] Example 3: A method for preparing mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0062] Step S1: inoculate and streak Bacillus subtilis and Bacillus licheniformis into beef extract peptone medium respectively, and culture at 37°C for 2 days; inoculate and streak lactic acid bacteria into MRS medium, and culture at 35°C for 5 days; inoculate and streak yeast into PDA medium, and culture at 30°C for 2 days; inoculate and streak activated Bacillus subtilis and Bacillus licheniformis into seed medium respectively, and culture at 37°C and 180r / min for 2 days to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid; inoculate and streak activated lactic acid bacteria into seed medium, and culture at 37°C and 180r / min for 3 days to form lactic acid bacteria seed liquid; inoculate and streak activated yeast into seed medium, and culture at 30°C and 180r / min for 3 days to form yeast seed liquid;

[0063] Beef extract peptone medium is: beef extract 5g / L, peptone 15g / L, sodium chloride 5g / L, agar 20g / L, pH=7.4, sterilized at 121℃ for 20min; MRS medium is: peptone 10g / L, beef extract 12g / L, yeast extract 5.0g / L, diammonium hydrogen citrate 4g / L, glucose 22g / L, Tween 80 2g / L, sodium acetate trihydrate 6g / L, dipotassium hydrogen phosphate trihydrate 4g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate monohydrate 0.28g / L, agar 20g / L, pH=6.5, sterilized at 121℃ for 20min; PDA medium is: peeled potato 202g / L, glucose 20g / L, agar 20g / L, sterilized at 121℃ for 20min;

[0064] The seed culture medium of Bacillus subtilis and Bacillus licheniformis is: beef extract 5g / L, peptone 15g / L, sodium chloride 5g / L, pH=7.4; the seed culture medium of lactic acid bacteria is: peptone 10g / L, beef extract 12g / L, yeast extract 5.0g / L, diammonium hydrogen citrate 4g / L, glucose 22g / L, Tween 80 2g / L, sodium acetate trihydrate 6g / L, dipotassium hydrogen phosphate trihydrate 4g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate monohydrate 0.28g / L, pH=6.5; the seed culture medium of yeast is: peeled potato 202g / L, glucose 20g / L;

[0065] The Bacillus subtilis seed liquid, the Bacillus licheniformis seed liquid, the lactic acid bacteria seed liquid, and the yeast seed liquid are uniformly mixed to obtain a fermentation mixed seed liquid (the mass ratio of each strain is: Bacillus subtilis: Bacillus licheniformis, lactic acid bacteria, yeast = 1:2:1.5:0.8);

[0066] Step S2: mixing the fermentation mixed seed liquid and the fermentation base material (the fermentation base material includes the following weight components: 50 parts of corn, 30 parts of soybean meal, 20 parts of fermented sweet potato residue, 1.5 parts of calcium hydrogen phosphate, 1.0 part of zeolite powder, and 0.5 parts of salt) at a mass ratio of 1:25 to obtain a mixed fermentation base material; then adding 4000U / g of immobilized complex enzyme and mixing evenly, adjusting the water content of the mixed fermentation base material to 50%, and fermenting at 30°C for 7 days to obtain a fermented mixed feed;

[0067] The fermented sweet potato residue was prepared by mixing 20 parts of sweet potato residue and 1 part of fermented mixed seed liquid, adding 1000U / g immobilized complex enzyme, and solid fermenting in a breathing bag for 65h.

[0068] The preparation method of the immobilized complex enzyme in step S2 is as follows:

[0069] Step (1): 4 parts of chitosan and 40 parts of hydrochloric acid solution are mixed evenly, 10 parts of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 4 parts of N-hydroxysuccinimide, and 4.8 parts of L-cysteine ​​are added, the pH value is adjusted to 6, the reaction is carried out for 5 hours, and after dialysis and freeze-drying, modified chitosan is obtained;

[0070] Step (2): 4 parts of modified chitosan and 1200 parts of deionized water are mixed evenly, the pH value of the system is adjusted to 4.4, and under the condition of 4000r / min high-speed shearing, 0.08 parts of 1.2mg / mL complex enzyme solution (the complex enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase, and xylanase according to the enzyme activity ratio of 1:2:1:1:0.6:10), 4.8 parts of zein and 720 parts of ethanol are added, and the mixture is stirred for 5h. After rotary evaporation, centrifugation and drying, an immobilized complex enzyme is obtained.

[0071] Comparative Example 1: A method for preparing mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0072] The fermented sweet potato residue is prepared by mixing 15 parts of sweet potato residue and 0.7 parts of fermentation mixed seed liquid, adding 200U / g immobilized complex enzyme, and solid fermentation in a breathing bag for 63 hours; compared with Example 2, Comparative Example 1 reduces the amount of immobilized complex enzyme added, and the other steps are the same as Example 2.

[0073] Comparative Example 2: A method for preparing mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0074] The fermented sweet potato residue is prepared by mixing 15 parts of sweet potato residue and 0.3 parts of fermentation mixed seed liquid, adding 800U / g immobilized complex enzyme, and solid fermentation in a breathing bag for 63 hours; compared with Example 2, Comparative Example 2 reduces the amount of fermentation mixed seed liquid added, and the other steps are the same as Example 2.

[0075] Comparative Example 3: A method for preparing mixed feed fermented by Bacillus licheniformis, comprising the following process:

[0076] The preparation method of the immobilized complex enzyme in step S2 is as follows:

[0077] 2 parts of chitosan and 550 parts of deionized water were mixed evenly, and the pH value of the system was adjusted to 4.2. Under the condition of 3500 r / min high-speed shearing, 0.06 parts of 0.5 mg / mL complex enzyme solution (the complex enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase, and xylanase according to the enzyme activity ratio of 1:1.5:1:1:0.55:9), 2.2 parts of zein and 300 parts of ethanol were added, and the mixture was stirred for 4 hours. After rotary evaporation, centrifugation and drying, the immobilized complex enzyme was obtained;

[0078] Compared with Example 2, in Comparative Example 3, the modified chitosan is replaced by chitosan; the other steps are the same as those in Example 2.

[0079] Comparative Example 4: A method for preparing mixed feed fermented by Bacillus licheniformis, comprising the following process

[0080] Compared with Example 2, in step (2) of Comparative Example 4, the composite enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase and xylanase in an enzyme activity ratio of 1:0.5:0.5:0.5:0.2:5; the other steps are the same as Example 2.

[0081] Experiment: The fermented sweet potato residues obtained in Examples 1-3 and Comparative Examples 1-4 and the sweet potato residues before fermentation (control group) were taken to prepare samples, and their properties were tested respectively and the test results were recorded:

[0082] The crude protein content was determined by the Kjeldahl method; the crude fiber content was determined by an automatic fiber analyzer (Gerhardt Fi-bretherm FT12, Germany); the energy value was determined by an isothermal automatic calorimeter (Changsha Kaiyuan Instrument 5E-AC8018); the lysine, threonine and methionine contents were determined by an automatic amino acid analyzer (Hitachi L-8800, Japan).

[0083] Test Results

[0084]

[0085] According to the data in the above table, we can clearly draw the following conclusions:

[0086] 1. Compared with Examples 1-3, the crude protein, lysine, threonine, methionine content and energy value of the fermented sweet potato residues prepared in Comparative Examples 1 and 2 were reduced, and the crude fiber content was increased, indicating that the performance of the fermented sweet potato residues prepared by the present invention is affected by the ratio of each reagent in the preparation process. Selecting the mass ratio within the said range can improve the nutrient level of the fermented sweet potato residues.

[0087] 2. Compared with Examples 1-3, the crude protein, lysine, threonine, methionine content and energy value of the fermented sweet potato residue prepared in Comparative Example 3 were reduced, and the crude fiber content was increased, which shows that compared with chitosan, the modified chitosan prepared by the present invention has better biocompatibility and stability, thereby improving the stability of the complex enzyme and further improving the fermentation effect of the sweet potato residue.

[0088] 3. Compared with Examples 1-3, the crude protein, lysine, threonine, methionine content and energy value of the fermented sweet potato residue prepared in Comparative Example 4 were reduced, and the crude fiber content was increased. It can be seen that the performance of the immobilized composite enzyme prepared by the present invention is affected by the ratio of each reagent in its preparation process. By selecting a mass ratio within the said range, the prepared immobilized composite enzyme has better performance, thereby improving the fermentation effect of the sweet potato residue.

[0089] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process method article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process method article or device.

[0090] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a mixed feed fermented by Bacillus licheniformis, characterized in that: The steps include: Step S1: activating Bacillus subtilis, Bacillus licheniformis, lactic acid bacteria, and yeast and inoculating them into seed culture medium for seed culture to obtain Bacillus subtilis seed solution, Bacillus licheniformis seed solution, lactic acid bacteria seed solution, and yeast seed solution; Evenly mixing the Bacillus subtilis seed liquid, the Bacillus licheniformis seed liquid, the lactic acid bacteria seed liquid, and the yeast seed liquid to obtain a fermentation mixed seed liquid; Step S2: Evenly mix the fermentation mixed seed liquid and the fermentation base material to obtain a mixed fermentation base material; then add the immobilized complex enzyme and mix evenly, adjust the water content of the mixed fermentation base material to 40-50%, and ferment at 20-30° C. for 5-7 days to obtain a fermented mixed feed.

2. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 1, characterized in that: The specific steps of seed culture in step S1 are: inoculating and streaking the activated Bacillus subtilis and Bacillus licheniformis into the seed culture medium respectively, and shaking and culturing them at 30-37° C. and 160-180 r / min for 1-2 days to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid; inoculating and streaking the activated lactic acid bacteria into the seed culture medium, and shaking and culturing them at 30-37° C. and 160-180 r / min for 2-3 days to form lactic acid bacteria seed liquid; inoculating and streaking the activated yeast into the seed culture medium, and shaking and culturing them at 28-30° C. and 160-180 r / min for 2-3 days to form yeast seed liquid.

3. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 1, characterized in that: The mass ratio of each bacterial species in the fermentation mixed seed solution in step S1 is: Bacillus subtilis: Bacillus licheniformis, lactic acid bacteria, yeast = 1: (1-2): (1.0-1.5): (0.5-0.8).

4. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 1, characterized in that: The total number of colonies in the fermented mixed seed solution in step S1 is 1×10 6 -1×10 8 CFU / g.

5. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 1, characterized in that: The preparation method of the immobilized complex enzyme in step S2 is as follows: Step (1): mixing chitosan and hydrochloric acid solution uniformly, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and then adding L-cysteine, adjusting the pH value to 5-6, reacting for 3-5 hours, dialyzing, and freeze-drying to obtain modified chitosan; Step (2): uniformly mix the modified chitosan and deionized water, adjust the pH value of the system to 4.0-4.4, add the mixed solution of the complex enzyme solution, zein and ethanol under the condition of high-speed shearing at 3000-4000 r / min, stir for 3-5 hours, and obtain the immobilized complex enzyme after rotary evaporation, centrifugation and drying.

6. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 5, characterized in that: In the step (2), the composite enzyme is prepared by compounding amylase, cellulase, β-mannanase, acid protease, pectinase and xylanase according to the enzyme activity ratio of 1:(1-2):1:1:(0.5-0.6):(8-10).

7. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 1, characterized in that: In step S2, the mass ratio of the fermentation mixed seed liquid to the fermentation base material is 1:(15-25).

8. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 8, characterized in that: The fermentation base material comprises the following components by weight: 40-50 parts of corn, 20-30 parts of soybean meal, 10-20 parts of fermented sweet potato residue, 1.0-1.5 parts of calcium hydrogen phosphate, 0.8-1.0 parts of zeolite powder, and 0.3-0.5 parts of salt.

9. The method for preparing a mixed feed fermented by Bacillus licheniformis according to claim 2, characterized in that: The fermented sweet potato residue is prepared by mixing sweet potato residue and fermented mixed seed liquid in a mass ratio of 1:(0.04-0.05), adding 500-1000U / g immobilized complex enzyme, and fermenting for 60-65h.

10. A mixed feed fermented by Bacillus licheniformis prepared according to the preparation method according to any one of claims 1 to 9.

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