Compound microbial fermentation feed and preparation method thereof

Abstract

The invention relates to the field of feed preparation, in particular to a compound microbial fermentation feed and a preparation method thereof. The method comprises the following steps: inoculating an activated compound microorganism strain into a fermentation culture medium taking agricultural solid wastes (corn sugar residues, bean residues and vinegar residues) as raw materials, and carrying out fermentation culture to obtain the compound microorganism fermented feed. According to the present invention, the non-grain biomass can be converted into the functional composite microbial fermentation feed with characteristics of no pollution, low cost, high safety, low three element content and high crude protein content by using the composite flora and combining the optimization of the fermentation conditions and the process, such that the comprehensive utilization of the agricultural and sideline processing waste is achieved, and the production cost is reduced. The value and the functionality of the non-grain biomass protein feed are greatly improved. The prepared compound microbial fermentation feed can be used for feeding animals, and has important strategic significance for promoting transformation and development of the feed industry.

Description

Technical Field

[0001] The invention relates to the field of feed preparation, and in particular to a composite microbial fermented feed and a preparation method thereof. Background Art

[0002] my country is a large livestock breeding country with a large grain import volume and a low implicit self-sufficiency rate. In 2023, my country's feed grain consumption accounted for about 53% of total grain consumption, and the demand for soybean meal for feed drove the import of soybeans and beans to 102.8 million tons. According to the current feed conversion level, it is estimated that my country's soybean and other protein feed gap will exceed 124 million tons in 2035. The demand for feed grain continues to grow due to the growth in demand for livestock products and structural changes. At the same time, grain consumption shows that the consumption of food grains decreases year by year, while the consumption of feed grains continues to rise.

[0003] At the same time, my country is also a large agricultural country, and a large amount of agricultural solid waste is generated every year. Among them, various types of wet bean dregs with high feasibility of application total about 25 million tons, and the output of fresh vinegar dregs is 3.68 million tons. According to incomplete survey statistics, the wet bean dregs in Nanjing, Yangzhou and Suzhou in Jiangsu Province alone are nearly 200,000 tons. Jiangsu vinegar dregs resources are also very rich, and the output of Zhenjiang Hengshun alone reaches 345,000 tons.

[0004] Therefore, in order to promote the conservation and consumption reduction of feed grain, ensure the stable supply of grain and important agricultural products, promote the development and utilization of non-grain biomass resources according to local conditions, and prepare new protein feed, it has become an important breakthrough to solve the problems of difficult processing of low-quality biomass and lack of feed protein.

[0005] However, it is difficult to handle agricultural and sideline processing waste, and the technology is backward and the equipment is simple. Usually, only a small amount is added as livestock and poultry feed, and the rest is mainly discarded or incinerated at will, causing resource waste and environmental pollution. Taking bean dregs and vinegar dregs, two agricultural and sideline processing wastes, as examples, their fiber accounts for more than 40% and 60% respectively. When used as roughage for feeding animals, they have low nutritional value and poor palatability; wet bean dregs have a high water content and are very easy to breed miscellaneous bacteria and even harmful bacteria. They are often prone to deterioration and taste, polluting the environment; at the same time, bean dregs also contain anti-nutritional factors, especially trypsin inhibitors, which will hinder the digestion and absorption of bean protein by trypsin in pigs, causing diarrhea and affecting growth. In addition, the acid substances remaining in vinegar dregs damage the gastric mucosa and small intestinal epithelial cells of animals. There is a pungent sour smell when they are piled outdoors, causing environmental pollution and other problems.

[0006] Therefore, it is of great practical significance to study new fermentation processes to convert non-food biomass into pollution-free, low-cost, and highly safe functional protein feed. In view of this, the present invention is proposed. Summary of the invention

[0007] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology, focus on the efficient conversion of non-grain protein feed resources, and conduct research on the exploration and breeding of edible fungi and probiotics for efficient enzymatic fiber and protein conversion, new green and efficient manufacturing processes for non-grain protein feed, and the safety and feeding effect evaluation of new protein feed products, thereby providing a composite microbial fermented feed.

[0008] Another technical problem to be solved by the present invention is to provide a method for preparing the composite microbial fermented feed.

[0009] Another technical problem to be solved by the present invention is to provide application of the composite microbial fermented feed.

[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0011] A composite microbial fermented feed, characterized in that the composite microbial fermented feed is prepared by using agricultural solid waste as raw materials and fermenting with composite microbial strains.

[0012] Wherein, the composite microbial strains are edible fungi, molds and yeasts.

[0013] Specifically, the edible fungi include any one or a combination of several of Ganoderma lucidum, Pleurotus eryngii, Pleurotus geesteranus, Oyster mushroom, Ganoderma lucidum, and Hericium erinaceus; the molds include any one or a combination of several of Phanerochaete chrysosporium, Trichoderma harzianum, Aspergillus niger, Trichoderma, Aspergillus niger, and Penicillium chrysogenum; and the yeasts include any one or a combination of several of Candida tropicalis, Pichia pastoris, Candida utilis, and Saccharomyces cerevisiae.

[0014] Preferably, the edible fungi are Pleurotus eryngii and / or Hericium erinaceus, the mold is any one or a combination of Trichoderma harzianum, Trichoderma, and Penicillium chrysogenum, and the yeast is any one or a combination of Candida tropicalis, Pichia pastoris, and Candida utilis.

[0015] More preferably, the edible fungus is Pleurotus eryngii, the mold is Trichoderma, and the yeast is Candida tropicalis.

[0016] In some embodiments of the present invention, the red oyster mushroom is GXGD-EF-13, which was donated by Jiangsu Academy of Agricultural Sciences; the Trichoderma is yz-682, which was purchased from the Agricultural Microbiology Center of China National Microbiological Culture Collection Committee, and the strain number is ACCC32582; the yeast is yz-1814, which was purchased from the Agricultural Microbiology Center of China National Microbiological Culture Collection Committee, and the strain number is ACCC 20275.

[0017] The agricultural solid waste includes any one or a combination of corn sugar residue, vinegar residue and bean dregs.

[0018] Preferably, the agricultural solid waste is corn sugar residue, vinegar residue and bean dregs.

[0019] A method for preparing composite microbial fermented feed comprises inoculating activated composite microbial strains into a fermentation medium using agricultural solid waste as raw materials for fermentation culture to obtain the composite microbial fermented feed.

[0020] The inoculation amount of each strain is 3-6% v / v (wherein the yeast cell concentration is 10 6 ~10 9 cfu / mL); the inoculation sequence includes any one of one-step inoculation, two-step inoculation and three-step inoculation.

[0021] Specifically,

[0022] (1) When the inoculation sequence is one-step inoculation: edible fungi, molds and yeasts are inoculated into the fermentation medium at the same time for fermentation culture;

[0023] (2) When the inoculation sequence is a two-step inoculation: the edible fungi and the mold are inoculated into the fermentation medium at the same time, and after an interval of 2 to 7 days, the yeast is inoculated for fermentation culture; or, the edible fungi and the yeast are inoculated into the fermentation medium at the same time, and after an interval of 2 to 7 days, the mold is inoculated for fermentation culture;

[0024] (3) When the inoculation sequence is a three-step inoculation: edible fungi, molds and yeasts are inoculated into the fermentation medium in sequence every 2 to 7 days for fermentation culture; that is, edible fungi are first inoculated into the fermentation medium, and after an interval of 2 to 7 days, molds are inoculated, and after an interval of 2 to 7 days, yeasts are finally inoculated into the fermentation medium for fermentation culture.

[0025] Preferably, the inoculation amount of each strain is 4-5% v / v (wherein the yeast cell concentration is 10 8 cfu / mL)

[0026] The preferred inoculation sequence is a two-step inoculation, in which the edible fungi and yeast are inoculated into the fermentation medium at the same time, and after an interval of 2 to 7 days, the remaining strains are inoculated for fermentation culture.

[0027] The culture conditions of the fermentation culture are as follows: the water content of the fermentation culture medium is controlled to be 40-60%, the fermentation temperature is 26-32° C., the initial pH value is 3-6, and the fermentation days are 3-8 days.

[0028] The preferred culture conditions are: controlling the water content of the fermentation medium to 45%, the fermentation temperature to 26° C., the initial pH value to 4-5, and fermentation for 6 days.

[0029] The fermentation medium has a formula of: 100-300 g / L corn sugar residue, 100-300 g / L vinegar dregs, 145-345 g / L bean dregs, 0.5-5 g / L organic nitrogen, 1-8 g / L inorganic nitrogen, 10-30 g / L carbon source, 1-10 g / L sodium chloride, and 1-10 g / L dipotassium hydrogen phosphate.

[0030] Specifically, the organic nitrogen is urea; the inorganic nitrogen includes any one or a combination of ammonium sulfate, ammonium carbonate and ammonium nitrate.

[0031] Preferably, the inorganic nitrogen is ammonium sulfate; the carbon source includes any one or a combination of glucose, brown sugar, and molasses, and the preferred carbon source is brown sugar; the ratio of ammonium sulfate to brown sugar in the fermentation medium is 1:4-5.

[0032] Preferably, the fermentation medium formula is: corn sugar residue 100g / L (dry matter), vinegar residue 200g / L (dry matter), bean dregs 200g / L (dry matter), urea 1g / L, ammonium sulfate 4.4g / L, brown sugar 20g / L, sodium chloride 5g / L, and potassium dihydrogen phosphate 5g / L.

[0033] The composite microbial fermented feed prepared by the above preparation method can effectively reduce the cellulose, hemicellulose and lignin content in the raw material, while the overall weight loss rate is also significantly reduced, and the crude protein content is increased. This shows that the composite bacterial community has a high ability to utilize inorganic nitrogen and reducing sugars. By utilizing the crude fiber in the raw material, bacterial protein is produced to reduce the crude fiber while also reducing the weight loss rate. The increase in crude protein content and the reduction in weight loss indicate that the quality of the generated bacterial protein makes up for the quality of the raw material consumption.

[0034] The application of the above-mentioned composite microbial fermented feed in feeding animals is also within the scope of protection of the present invention.

[0035] Wherein, the animals are beef cattle and / or fattening pigs.

[0036] Beneficial effects:

[0037] (1) The present invention can transform non-grain biomass (corn sugar residue, bean dregs and vinegar residue) into pollution-free, low-cost and highly safe functional composite microbial fermented feed through a new fermentation process, thereby realizing the comprehensive utilization of agricultural and sideline processing waste and greatly improving the value and functionality of non-grain biomass protein feed.

[0038] (2) The present invention analyzes the characteristics of three raw materials, namely corn molasses, bean dregs and vinegar dregs, and adaptively conducts bacterial species mining to obtain a composite bacterial community with high ability to utilize inorganic nitrogen and reducing sugars. The composite microbial fermentation feed prepared by the composite bacterial community, combined with the optimization of fermentation conditions and processes, can effectively reduce the cellulose, hemicellulose and lignin content in non-food biomass (corn molasses, bean dregs and vinegar dregs), significantly reduce the overall weight loss rate, and at the same time increase the crude protein content in the feed.

[0039] (3) The composite microbial fermented feed prepared by the present invention can be used to feed animals, which has great strategic significance for promoting the transformation and development of the feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0041] Figure 1 This is the antagonism between edible fungi and mold.

[0042] Figure 2 This is the consumption of nitrogen in the supernatant by yeast after nitrogen acclimation.

[0043] Figure 3 Shows the biomass of yeast in different generations after nitrogen acclimation.

[0044] Figure 4 This is the consumption of reducing sugars in the supernatant by yeast after carbon acclimation.

[0045] Figure 5 Fermentation of yeast before and after acclimation.

[0046] Figure 6 This is the status of the three elements (cellulose, hemicellulose, and lignin) after yeast fermentation before and after domestication.

[0047] Figure 7 The effect of different inoculation sequences on fermentation results.

[0048] Figure 8 It is the fermentation result under the optimal formula and process. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0050] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0051] In the following examples, the sources of the 24 strains are shown in Table 1.

[0052] Table 1 Sources and names of 24 bacterial strains

[0053]

[0054] Note: 1. Donated by Kunming Edible Fungi Research Institute

[0055] 2. Donated by Jiangsu Academy of Agricultural Sciences

[0056] 3. Unit procurement (Lijiang Ancient Town Farmers Market)

[0057] 4. Nanjing Liuhe Suguo Supermarket

[0058] 5. Agricultural Microbiology Center of China Microbiological Culture Collection Administration

[0059] 6. Laboratory self-isolation

[0060] Example 1 Preliminary screening of strains suitable for corn sugar residue, vinegar residue and bean dregs

[0061] Nine large edible fungi, seven molds and eight yeasts were selected from the company's fungus library (specific strain information is shown in Table 1). After the edible fungi were activated with edible fungi mother culture medium, the molds and yeasts were activated with modified PDA solid / liquid culture medium, respectively. Then, the edible fungi blocks that grew vigorously on the edible fungi mother culture medium, the mold blocks and yeast liquid that grew vigorously on the modified PDA were selected. Specifically, yeasts and molds were cut into 6mm×6mm blocks according to the punching method and inoculated into solid culture media with different by-product raw materials (corn sugar residue, vinegar residue or bean residue) as the only carbon source and nitrogen source, and cultured at 26°C until the hyphae covered the entire plate, and the time required for the hyphae to cover the entire plate was calculated. The yeast was cultured by the plate streak method, with the same bacterial concentration (10 8 cfu / mL) on a plate, and evenly spread it on a solid culture medium with different byproduct raw materials (corn sugar residue, vinegar residue or bean dregs) as the only carbon and nitrogen source, and culture it at 30°C until colonies appear, and count the growth cycle of yeast at the same bacterial concentration. In this way, the edible fungi, molds and yeasts with the fastest growth rate in corn sugar residue, vinegar residue and bean dregs can be screened out.

[0062] The formula of the edible fungus mother culture medium is: 20% potato boiled juice, 20g sucrose, 20g agar, add water to 1000mL. Specifically, wash and peel the potatoes, and cut them into small pieces. Weigh 200g of potato pieces, add 1000mL of water, boil for 20 minutes, and filter. Add water to the filtered juice to 1000mL to make 20% potato boiled juice. Add agar and sucrose to the potato boiled juice, boil it, dissolve it, add water, package it, sterilize it, and set it aside. The use of this culture medium does not have strict requirements on the pH value and does not need to be measured.

[0063] The solid culture medium is formulated as follows: 4% byproduct raw materials (corn sugar residue, vinegar residue or bean residue), 2% agar, and the raw materials are crushed and passed through a 50-mesh sieve before use.

[0064] The modified PDA liquid culture medium formula is: 200g / L potato, 20g / L glucose, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate, VB 1 0.1g / L, natural pH. If 2% agar is added, it becomes a modified PDA solid culture medium formula.

[0065] The results of the full-plate growth cycles of different edible fungi, molds and yeasts on the screening plates are shown in Table 2. Analysis of the data in Table 2 shows that the growth cycles of the top three edible fungi are ranked from small to large: GXGD-EF-13>GXGD-EF-25>GXGD-EF-72; the growth rates of the top three molds in the screening plates are: yz-675>yz-777>yz-682; the growth rates of the top three yeasts in the screening plates are: yz-391=yz-1720>yz-1814>yz-390.

[0066] Table 2 The period of full growth of 24 strains on the screening plate

[0067]

[0068] Example 2 Screening of high enzyme activity and high protein strains

[0069] The 10 strains screened in Example 1 were inoculated into liquid culture medium for edible fungi, and molds and yeasts were inoculated into modified PDA liquid culture medium, wherein the edible fungi were cultured for 4 to 6 days until the size of the balls was uniform, the molds were cultured for 1 to 2 days, and the yeasts were cultured for 1 to 2 days. The cultured samples were centrifuged, and the supernatant was used to detect cellulase activity, hemicellulase activity, and laccase activity. The crude protein content was detected after the solids were dried. Among them, crude protein was detected according to the method in GB / T6432-2018 "Determination of crude protein in feed by Kjeldahl nitrogen determination"; cellulase activity was determined according to the method in GB / T23881-2009 "Filter paper method for determination of feed cellulase activity"; hemicellulase activity and laccase activity were carried out according to the method in DB41 / T 1615-2018 "Technical regulations for detection of edible fungi mycelium cellulase, xylanase, laccase, and protease activity".

[0070] The formula of the edible fungus mother culture medium is: 20% potato boiled juice, 20g / L sucrose, and natural pH.

[0071] The formula of the modified PDA liquid culture medium is: 200g / L potato, 20g / L glucose, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate, VB 1 0.1g / L, pH natural.

[0072] Table 3 Enzyme activity and crude protein content of different strains

[0073]

[0074]

[0075] According to the data in Table 3, among the three edible fungi, the enzyme activities of cellulose and hemicellulose in GXGD-EF-13 and GXGD-EF-25 are relatively high. Combined with the growth rates of these two strains, GXGD-EF-13 grows faster. Among the three molds, yz-682 has the highest laccase activity, and the cellulose and hemicellulose enzyme activities are relatively high; yz-777 has the highest hemicellulose enzyme activity, but yz-777 will produce antibiotics during the fermentation process of yellow Penicillium, so this type of fungus is generally not used in feed. When selecting the target strains of edible fungi and molds, it is also necessary to determine the corresponding target strains based on the antagonism between edible fungi and molds. Among the four yeast strains, yz-1814 has the highest crude protein content, so yz-1814 is selected as the target strain.

[0076] Example 3 Antagonism test between edible fungi and mold

[0077] Use a 5mm diameter Oxford cup to cut GXGD-EF-13, GXGD-EF-25, yz-460, yz-675, yz-680, yz-682, yz-684, yz-777, QM-2 from the culture medium of edible fungi and molds, transfer the mycelium downward to the center of the PDA culture medium plate (9cm diameter), and culture at 26℃ for 5 days. Observe the coverage area between each pair. Figure 1 As can be seen from Table 4, GXGD-EF-13, GXGD-EF-25 and three strains of Aspergillus niger (yz-680, yz-684, QM-2) have obvious antagonistic lines, indicating that Aspergillus niger and edible fungi are not suitable for growing at the same time. The antagonistic photos of GXGD-EF-13 and two strains of Trichoderma (yz-675 and yz-682) show that GXGD-EF-13 and yz-675 have antagonism but it is relatively weak, and there is no antagonism between GXGD-EF-13 and yz-682, and these two strains can coexist. GXGD-EF-25 has antagonism with two strains of Trichoderma (yz-675 and yz-682) but it is relatively weak. The antagonistic photos of GXGD-EF-13, GXGD-EF-25 and yz-777 show that GXGD-EF-13, GXGD-EF-25 and yz-777 have no antagonism and can coexist with each other. Combined with the enzyme activities of edible fungi and molds in Example 2, GXGD-EF-13 and yz-682 were finally selected as the target strains for fermentation.

[0078] Table 4 Antagonism between edible fungi and molds

[0079] Edible Fungi Mold Antagonism / symbiosis GXGD-EF-13 yz-460 Antagonism GXGD-EF-13 yz-680 Antagonism GXGD-EF-13 yz-684 Antagonism GXGD-EF-13 QM-2 Antagonism GXGD-EF-13 yz-675 Antagonism GXGD-EF-13 yz-682 Symbiosis GXGD-EF-13 yz-777 Symbiosis GXGD-EF-25 yz-460 Antagonism GXGD-EF-25 yz-680 Antagonism GXGD-EF-25 yz-684 Antagonism GXGD-EF-25 QM-2 Antagonism GXGD-EF-25 yz-675 Antagonism GXGD-EF-25 yz-682 Antagonism GXGD-EF-25 yz-777 Symbiosis

[0080] Example 4 Carbon and nitrogen acclimation of yeast

[0081] 1. Yeast nitrogen acclimation

[0082] When the carbon, nitrogen and inorganic salt content of the culture medium is the same, the ratio of organic nitrogen (urea) to inorganic nitrogen (ammonium sulfate) is adjusted to prepare a solid selective culture medium with urea and ammonium sulfate as the only nitrogen sources, and the growth of yeast is investigated. After the amount of urea added to the culture medium is determined, an acclimation test is conducted on the yeast with increasing amounts of ammonium sulfate added.

[0083] Formula 1: urea 1g / L, ammonium sulfate 3g / L, brown sugar 30g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L;

[0084] Formula 2: 0.5g / L urea, 4g / L ammonium sulfate, 30g / L brown sugar, 5g / L sodium chloride, 5g / L potassium dihydrogen phosphate;

[0085] Formula 3: urea 2.5g / L, ammonium sulfate 0g / L, brown sugar 30g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L;

[0086] The specific nitrogen content information of formulas 1 to 3 is shown in Table 5.

[0087] Table 5 Specific nitrogen content information of formula 1 to 3

[0088]

[0089] The same concentration of yeast yz-1814 was inoculated into the above three formulations. It was found that yz-1814 grew fastest in formulation 1, so ammonium sulfate in formulation 1 was selected as inorganic nitrogen. The carbon source content in the nitrogen acclimation liquid medium was fixed, and the ammonium sulfate content was gradually increased. The initial ammonium sulfate content was 3g / L, and the ammonium sulfate content was increased every 3 generations with a gradient of 10%. After acclimation to the 15th generation, the final ammonium sulfate content was 4.4g / L and the total nitrogen content was 98g / 100g.

[0090] Nitrogen acclimation liquid medium (1st to 3rd generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0091] Nitrogen acclimation liquid medium (4-6 generations): urea 1g / L, ammonium sulfate 3.3g / L, brown sugar 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0092] Nitrogen acclimation liquid medium (7-9 generations): urea 1g / L, ammonium sulfate 3.63g / L, brown sugar 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0093] Nitrogen acclimation liquid medium (10-12 generations): urea 1g / L, ammonium sulfate 4g / L, brown sugar 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0094] Nitrogen acclimation liquid culture medium (13-15 generations): urea 1g / L, ammonium sulfate 4.4g / L, brown sugar 30g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L.

[0095] from Figure 2 and Figure 3 From the data in the results, it can be seen that after 13 to 15 generations of domestication, the utilization rate of inorganic nitrogen in the supernatant by yeast yz-1814 increased by 43.9 to 53% compared with the blank group; Figure 3 The data in the results show that the biomass of the domesticated bacteria also increased to 1.8-2.1 g / L. The above results show that the domesticated bacteria have a higher ability to utilize inorganic nitrogen and improve the efficiency of converting inorganic nitrogen to synthesize microbial protein.

[0096] 2. Carbon acclimation of yeast

[0097] When the carbon, nitrogen and inorganic salt content of the culture medium is the same, different carbon sources (glucose, brown sugar, molasses) are selected to prepare solid selective culture medium with these carbon sources as the sole carbon source. The growth of yeast is investigated, and after the type of carbon source in the culture medium is determined, the yeast is subjected to an acclimation test with a reduced amount of carbon source added.

[0098] Formula 1: urea 1g / L, ammonium sulfate 3g / L, glucose 25g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L;

[0099] Formula 2: urea 1g / L, ammonium sulfate 3g / L, brown sugar 50g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L;

[0100] Formula 3: urea 1g / L, ammonium sulfate 3g / L, molasses 75g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L.

[0101] The specific carbon content information of formulas 1 to 3 is shown in Table 6.

[0102] Table 6 Specific carbon content information of formula 1 to 3

[0103] Total sugar (%) Addition amount (g / L) Carbon content (%) glucose 100 25 1000 brown sugar 50 50 1000 Molasses 33.3 75 999

[0104] The same concentration of yeast yz-1814 was inoculated into the above three recipes. It was found that yz-1814 grew fastest in recipe 2, so brown sugar was selected as the carbon source in recipe 2. The nitrogen source (ammonium sulfate) in the carbon acclimation liquid medium was fixed, and the brown sugar content was reduced by 10% every 3 generations, and finally reduced to 20g / L, and a total of 15 generations were subcultured.

[0105] Carbon acclimation liquid medium (1st to 3rd generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 50g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0106] Carbon acclimation liquid medium (4-6 generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 42.5g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0107] Carbon acclimation liquid medium (7-9 generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 35g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0108] Carbon acclimation liquid medium (10-12 generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 27.5g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L;

[0109] Carbon acclimation liquid culture medium (13-15 generations): urea 1g / L, ammonium sulfate 3g / L, brown sugar 20g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L.

[0110] from Figure 4 According to the data, after 13 to 15 generations of domestication, the utilization rate of reducing sugar in the supernatant by the domesticated bacteria increased by 97 to 98.3% compared with the blank group. This shows that the bacteria gradually reduce their dependence on reducing sugar and adapt to the low reducing sugar environment. The bacteria grown in this environment are more adaptable to high-fiber raw materials such as corn sugar residue, vinegar residue, and bean residue, thereby stimulating and inducing the bacteria to produce enzymes to utilize the wood fiber components in the raw materials, thereby reducing the three elements (cellulose, hemicellulose and lignin).

[0111] In summary, according to the final ratio of carbon acclimation and nitrogen acclimation in the culture medium, the ratio of ammonium sulfate and brown sugar in the auxiliary materials was determined to be 1:4.5.

[0112] Example 5 Characterization of domesticated yeast in fermentation medium containing by-product raw materials

[0113] The untamed and tamed yeast YZ-1814 were mixed at 10-20% v / v (the bacterial concentration was 10 6 ~10 9 cfu / mL) are inoculated into the fermentation medium. The fermentation medium formula is: corn sugar residue 100-300g / L (dry matter), vinegar residue 100-300g / L (dry matter), bean dregs 145-345g / L (dry matter), urea 0.5-5g / L, ammonium sulfate 1-8g / L, brown sugar 10-30g / L, sodium chloride 1-10g / L, potassium dihydrogen phosphate 1-10g / L. The water content of the fermentation medium is controlled to be 40-60%, the fermentation temperature is 26-32°C, the initial pH value is 4-6, and the fermentation is carried out for 3-8 days.

[0114] The untamed and tamed yeast YZ-1814 were cultured at 15% v / v (bacterial concentration was 10 8 cfu / mL) were inoculated into the fermentation medium. The fermentation medium formula is: corn sugar residue 100g / L (dry matter), vinegar residue 200g / L (dry matter), bean dregs 200g / L (dry matter), urea 1g / L, ammonium sulfate 4.4g / L, brown sugar 20g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L. The moisture content of the fermentation medium was controlled to be 45%, the fermentation temperature was 30°C, the initial pH value was 4, and the fermentation was carried out for 6 days.

[0115] from Figure 5According to the data, the crude protein content of the domesticated yeast after fermentation reached 30.21%, which was 36.6% higher than that before domestication; the nitrogen consumption rate reached 53.88%, which was 98.0% higher than that before domestication; the sugar consumption rate reached 94.63%, which was 5.2% higher than that before domestication; and the weight loss rate reached 1.89%, which was 89.73% lower than that before domestication. This shows that the domesticated bacteria have a high ability to utilize inorganic nitrogen and reducing sugars. The increase in crude protein content and the decrease in weight loss indicate that the quality of the generated bacterial protein makes up for the quality of the raw material consumption.

[0116] from Figure 6 The data analysis shows that after fermentation by domesticated yeast, the contents of the three elements are reduced. Among them, the cellulose content is reduced to 18.91%, a decrease of 53.22%; hemicellulose and lignin are reduced to 11.24% and 16.43%, respectively, a decrease of 27.62% and 22.21%. The test results show that the domesticated yeast has a high degree of degradation of the three elements.

[0117] Example 6 Fermentation process optimization

[0118] From the growth cycles of edible fungi, molds and yeasts in Example 1, it can be seen that edible fungi grow slowly on solid culture media prepared from three different by-product raw materials (corn sugar residue, vinegar residue or bean dregs), so the first step in this example is to inoculate edible fungi.

[0119] Method 1: The inoculation is divided into three steps, and the edible fungus DXGD-EF-13, mold yz-682, and yeast yz-1814 are inoculated into the fermentation medium in turn. The inoculation amount is 3-6% v / v, and the inoculation time interval is 2-7 days. The fermentation conditions are: the moisture content is controlled at 40-60%, the fermentation temperature is 26-32°C, the initial pH value is 4-6, and the fermentation is 3-8 days.

[0120] Method 2: The inoculation is divided into two steps. First, the edible fungus DXGD-EF-13 and the yeast yz-1814 are inoculated at the same time, with the inoculation amount being 3-6% v / v respectively. After an inoculation time interval of 2-7 days, the mold yz-682 is inoculated at an inoculation amount of 3-6% v / v. The culture is carried out for 2-7 days. The fermentation conditions are: the moisture content is controlled at 40-60%, the fermentation temperature is 26-32°C, the initial pH value is 4-6, and the fermentation is carried out for 3-8 days.

[0121] Method 3: The inoculation is divided into two steps. First, the edible fungus DXGD-EF-13 and the mold yz-682 are inoculated at a rate of 3-6% v / v each, and the inoculation time interval is 2-7 days. Then, the yeast yz-1814 is inoculated at a rate of 3-6% v / v. The culture is carried out for 2-7 days. The fermentation conditions are as follows: the moisture content is controlled at 40-60%, the fermentation temperature is 26-32°C, the initial pH value is 4-6, and the fermentation is carried out for 3-8 days.

[0122] Method 4: Direct one-step inoculation, the bacterial liquid of edible fungus DXGD-EF-13, mold yz-682 and yeast yz-1814 are inoculated into the fermentation medium at the same time, the inoculation amount is 3-6% v / v, the culture is carried out for 3-7 days, the fermentation conditions are: control the moisture content at 40-60%, the fermentation temperature at 26-32°C, the initial pH value at 4-6, and the fermentation is carried out for 3-8 days.

[0123] The fermentation medium formula is: corn sugar residue 100-300 g / L (dry matter), vinegar lees 100-300 g / L (dry matter), bean dregs 145-345 g / L (dry matter), urea 0.5-5 g / L, ammonium sulfate 1-8 g / L, brown sugar 10-30 g / L, sodium chloride 1-10 g / L, and dipotassium hydrogen phosphate 1-10 g / L.

[0124] The preferred fermentation medium formula is: corn sugar residue 100g / L (dry matter), vinegar residue 200g / L (dry matter), bean dregs 200g / L (dry matter), urea 1g / L, ammonium sulfate 4.4g / L, brown sugar 20g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 5g / L; fermentation conditions: the inoculation amount of each strain is 4% v / v (the cell concentration of yeast is 10 8 cfu / mL), the water content of the fermentation medium was controlled at 45%, the fermentation temperature was 26°C, the initial pH value was 4, and the fermentation was carried out for 6 days.

[0125] After the cultivation was completed, the samples obtained by the four methods were dried, and the contents of crude protein and three elements in the samples were tested to compare the effects of different inoculation sequences on the fermentation results. Figure 7 From the data analysis, it can be seen that in method 2, the crude protein content is the highest at 30.21%, and the three elements also decrease the most. This shows that the inoculation of edible fungi and yeast first is conducive to the initial degradation of raw materials by edible fungi, and then the inoculation of molds further increases the degradation of raw materials by the flora. At the same time, yeast can cover up the musty smell of molds, making the product full of yeast aroma.

[0126] Example 7 Optimization of fermentation conditions

[0127] The optimal inoculation order of three different types of bacteria obtained by Example 6 is: first inoculate edible fungi DXGD-EF-13 and yeast yz-1814, and finally inoculate mold yz-682. This example further optimizes the initial pH, water content and inoculation amount of the fermentation conditions by an orthogonal test method, with crude protein as the investigation index. The fermentation process refers to Example 6 Method 2. The specific fermentation condition optimization parameters are shown in Table 7, and the optimization results are shown in Table 8.

[0128] Table 7 Optimization parameters of fermentation conditions

[0129]

[0130] Table 8 Fermentation condition optimization results

[0131]

[0132] According to the results of the orthogonal test, the optimal group is the A3B2C2 group, that is, 15% v / v total inoculation amount, 45% water content and the initial pH value adjusted to 4.

[0133] Example 8 Optimal Formula and Process

[0134] Fermentation formula: corn sugar residue 100-300g / L (dry matter), vinegar dregs 100-300g / L (dry matter), bean dregs 145-345g / L (dry matter), urea 1g / L, ammonium sulfate 4.4g / L, brown sugar 20g / L, sodium chloride 5g / L, dipotassium hydrogen phosphate 5g / L.

[0135] Fermentation conditions: 45% water content, initial pH value adjusted to 4, 26°C fermentation temperature, and culture for 3 to 8 days.

[0136] Inoculation sequence: 15% v / v total inoculation amount, first inoculate edible fungi and yeast, with an inoculation amount of 5% each, and then inoculate mold at an inoculation amount of 5% after an inoculation time interval of 2 to 7 days.

[0137] according to Figure 8 The fermentation results show that after fermentation according to this formula and process, the crude protein of the fermentation product can reach 31.94%, and the cellulose, hemicellulose and lignin are also greatly reduced (17.43%, 11.81% and 13.36% respectively), and the overall weight loss rate after fermentation is also reduced (4.81%), indicating that the composite flora utilizes the crude fiber in the raw material, produces bacterial protein, reduces the crude fiber and also reduces the weight loss rate. The fermentation product prepared by this method can be used as a composite microbial fermentation feed for beef cattle and fattening pigs, thereby playing a role in partially replacing soybean dregs protein.

[0138] The present invention provides a composite microbial fermented feed and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A composite microbial fermented feed, characterized in that: The composite microbial fermented feed is made from agricultural solid waste as raw material and is fermented by composite microbial strains; Wherein, the composite microbial strains are edible fungi, molds and yeasts; Among them, the edible fungi include any one or a combination of several of Ganoderma lucidum, Pleurotus eryngii, Pleurotus geesteranus, Oyster mushroom, Ganoderma lucidum, and Hericium erinaceus; the molds include any one or a combination of several of Phanerochaete chrysosporium, Trichoderma harzianum, Aspergillus niger, Trichoderma, Aspergillus niger, and Penicillium chrysogenum; and the yeasts include any one or a combination of several of Candida tropicalis, Pichia pastoris, Candida utilis, and Saccharomyces cerevisiae.

2. The composite microbial fermented feed according to claim 1, characterized in that: The agricultural solid waste includes any one or a combination of corn sugar residue, vinegar residue and bean residue.

3. The composite microbial fermented feed according to claim 1, characterized in that: The edible fungi are red oyster mushrooms and / or Hericium erinaceus, the mold is any one of Trichoderma harzianum, Trichoderma, and Penicillium chrysogenum or a combination of several of them, and the yeast is any one of Candida tropicalis, Pichia pastoris, and Candida utilis or a combination of several of them.

4. The composite microbial fermented feed according to claim 1, characterized in that: The edible fungus is Pleurotus eryngii, the mold is Trichoderma, and the yeast is Candida tropicalis.

5. The method for preparing the composite microbial fermented feed according to claim 1, characterized in that: The activated composite microorganism strain is inoculated into a fermentation medium using agricultural solid waste as raw material for fermentation culture to obtain the composite microorganism fermented feed.

6. The preparation method according to claim 5, characterized in that: The inoculation, wherein the inoculation amount of each strain is 3-6% v / v; the inoculation sequence includes any one of one-step inoculation, two-step inoculation and three-step inoculation; (1) When the inoculation sequence is one-step inoculation: edible fungi, molds and yeasts are inoculated into the fermentation medium at the same time for fermentation culture; (2) When the inoculation sequence is a two-step inoculation: the edible fungi and the mold are inoculated into the fermentation medium at the same time, and after an interval of 2 to 7 days, the yeast is inoculated for fermentation culture; or, the edible fungi and the yeast are inoculated into the fermentation medium at the same time, and after an interval of 2 to 7 days, the mold is inoculated for fermentation culture; (3) When the inoculation sequence is a three-step inoculation: edible fungi, molds and yeasts are inoculated into the fermentation medium in sequence every 2 to 7 days for fermentation culture.

7. The preparation method according to claim 5, characterized in that: The fermentation culture has the following culture conditions: the water content of the fermentation culture medium is controlled to be 40-60%, the fermentation temperature is 26-32° C., the initial pH value is 3-6, and the fermentation days are 3-8 days.

8. The preparation method according to claim 5, characterized in that: The fermentation medium has a formula of: 100-300 g / L corn sugar residue, 100-300 g / L vinegar residue, 145-345 g / L bean dregs, 0.5-5 g / L organic nitrogen, 1-8 g / L inorganic nitrogen, 10-30 g / L carbon source, 1-10 g / L sodium chloride, and 1-10 g / L dipotassium hydrogen phosphate.

9. The preparation method according to claim 5, characterized in that: The organic nitrogen is urea; the inorganic nitrogen includes any one or a combination of ammonium sulfate, ammonium carbonate and ammonium nitrate, and the preferred inorganic nitrogen is ammonium sulfate; the carbon source includes any one or a combination of glucose, brown sugar and molasses, and the preferred carbon source is brown sugar; the ratio of ammonium sulfate to brown sugar in the fermentation medium is 1:4-5.

10. Use of the composite microbial fermented feed according to any one of claims 1 to 4 in feeding animals, wherein: The animals are beef cattle and / or fattening pigs.

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