A method for producing protein feed by two-stage solid fermentation of microbial corn skin

Abstract

This invention discloses a method for producing protein feed from sprayed corn husks via two-stage solid-state fermentation using microorganisms. The protein feed is obtained by two-stage solid-state fermentation of sprayed corn husks and urea using a compound microorganism. The compound microorganisms include Aspergillus niger, Saccharomyces cerevisiae, Bacillus licheniformis, and Pediococcus pentosaceus. The two-stage solid-state fermentation includes two stages: aerobic solid-state fermentation and anaerobic solid-state fermentation. The crude protein content of the obtained sprayed corn husk protein feed product is 29%–36%, of which the nitrogen-converted protein content of true protein, peptides, and amino acids is 23%–29%, while the nitrogen-converted protein content of urea and inorganic ammonia nitrogen is only 3%–6%. The feed fermentation process can effectively convert low-cost urea into high-value protein, amino acids, and peptides that can be utilized by animals, thereby reducing the cost of protein production.

Description

Technical Field

[0001] This invention relates to the field of biological feed, specifically to a method for producing protein feed from corn husks through a two-stage solid-state fermentation process using microorganisms. Background Technology

[0002] In recent years, with the continuous expansion of my country's livestock industry, a severe shortage of feed protein resources has emerged. Using biotechnology to treat waste residue, waste materials, and waste liquids from agricultural and sideline product processing to produce bio-feed or additives can not only reduce the environmental pollution caused by these waste biomass materials and achieve high-value utilization of waste resources, but also replace soybean meal, corn, and other feed ingredients in livestock farming, alleviating the current shortage of feed protein and promoting the green and sustainable development of my country's livestock industry.

[0003] Spray-processed corn husks are a byproduct of wet corn processing, produced by spraying corn slurry onto corn husks and then drying them. my country's current annual production is approximately 429,000 tons. Spray-processed corn husks with a crude protein content exceeding 15% and a crude fiber content below 15% can be directly fed as animal feed, with a market price of approximately 1500 yuan / ton. However, spray-processed corn husks suffer from an unbalanced nutritional structure, leading to complex feeding processes and low digestibility. Furthermore, during storage, spray-processed corn husks are prone to high levels of mycotoxins and decreased palatability, directly inhibiting their application in the feed industry. Although a Chinese patent application (application number 2018115725454) describes fermenting spray-processed corn husks, it only improves the number of probiotics and the amino acid structure of the feed product; the true protein content decreases from 15% to 10.5%, a 30% reduction compared to before fermentation, resulting in protein loss.

[0004] Bio-fermentation technology can be used to convert inexpensive non-protein nitrogen using feed microorganisms, which can effectively increase the protein content and value of corn husks. However, currently, the utilization rate of non-protein nitrogen urea by microorganisms (such as Saccharomyces cerevisiae, Bacillus, and Lactic acid bacteria) is generally only 30%. Therefore, selecting microorganisms or combinations of microorganisms with the ability to assimilate inexpensive non-protein nitrogen (such as urea) is of great significance for the efficient production of protein feed.

[0005] In addition, bio-fermentation can effectively reduce the crude fiber content in sprayed corn husks, while improving their palatability and probiotic content, lowering the pH value, avoiding mold contamination, and extending the shelf life. This is of positive significance for increasing the usage and effectiveness of sprayed corn husks in feed, enhancing their economic value, and addressing feed shortages. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for producing protein feed from corn husks by two-stage solid-state fermentation of microorganisms, which addresses the shortcomings of the existing technology.

[0007] To address the aforementioned technical problems, this invention discloses a method for producing protein feed from corn husks through a two-stage solid-state fermentation process using microorganisms with urea assimilation capabilities. The specific technical solution is as follows:

[0008] A method for producing protein feed from corn husks by two-stage solid-state fermentation of microorganisms includes the following steps: inoculating aerobic bacterial liquid into fermentation raw materials for aerobic solid-state fermentation to obtain aerobic fermentation products; then inoculating anaerobic bacterial liquid into the aerobic fermentation products for anaerobic solid-state fermentation to obtain protein feed.

[0009] The aerobic bacterial solution includes Aspergillus niger solution, Saccharomyces cerevisiae solution, and Bacillus licheniformis solution; the Aspergillus niger and Saccharomyces cerevisiae are microorganisms with urea assimilation capabilities.

[0010] The anaerobic bacterial solution includes Pediococcus pentosaceus bacterial solution.

[0011] The *Aspergillus niger* is ATCC 16888; the *Saccharomyces cerevisiae* is ATCC 18824; the *Bacillus licheniformis* is ATCC 14580; and the *Pediococcus pentosaceus* is ATCC 33316.

[0012] The viable count of Aspergillus niger in the Aspergillus niger solution is 0.5–2 × 10⁻⁶. 7 CFU / g; the viable count of Saccharomyces cerevisiae in the Saccharomyces cerevisiae broth is 4–6 × 10⁻⁶. 6 CFU / g; the viable count of Bacillus licheniformis in the bacterial culture is 0.1–1.0 × 10⁻⁶ CFU / g. 6 CFU / g; the viable count of *Pediococcus pentosaceus* in the bacterial suspension is 0.1–1.0 × 10⁻⁶ CFU / g. 6 CFU / g. Preferably, the viable count of Aspergillus niger in the Aspergillus niger solution is 1.0 × 10⁻⁶. 7 CFU / g; the viable count of Saccharomyces cerevisiae in the Saccharomyces cerevisiae broth is 5.0 × 10⁻⁶. 6 CFU / g; the viable count of Bacillus licheniformis in the bacterial culture was 0.5 × 10⁻⁶. 6 CFU / g; the viable count of *Pediococcus pentosaceus* in the bacterial culture was 0.5 × 10⁻⁶ CFU / g. 6 CFU / g.

[0013] The volume ratio of the Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid, and Pediococcus pentosaceus liquid is 1-2:1-2:0.5-1:0.5-1. Preferably, the volume ratio of the Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid, and Pediococcus pentosaceus liquid is 2:2:1:1.

[0014] The fermentation raw materials include dry matter, which comprises sprayed corn husks and urea, wherein the mass ratio of urea in the dry matter is 1% to 5%; preferably, the mass ratio of urea in the dry matter is 3.63%. Preferably, the sprayed corn husks are sprayed corn husks that have been crushed and passed through a 40-mesh round hole sieve, and sterilized at 115-121℃ after adding water. After sterilization, urea is added to obtain the fermentation raw materials.

[0015] The dry matter accounts for 10% to 80% of the total fermentation mass, preferably 25% to 45%, and more preferably 35%. The total fermentation mass refers to the total mass of the three solutions: fermentation raw materials, aerobic bacterial solution, and anaerobic bacterial solution.

[0016] The mass ratio of the aerobic bacterial solution to dry matter is 1% to 50%, preferably 2% to 38%, more preferably 14% to 32%, and even more preferably 20%.

[0017] The aerobic solid-state fermentation is carried out at a temperature of 15–50°C for a fermentation time of 1–336 hours; the anaerobic solid-state fermentation is carried out at a temperature of 20–40°C for a fermentation time of 1–72 hours. Preferably, the aerobic solid-state fermentation is carried out at a temperature of 22–44°C for a fermentation time of 72–168 hours, more preferably at 36°C for 72 hours; the anaerobic solid-state fermentation is carried out at a temperature of 36°C for 48 hours.

[0018] Secondly, the present invention also provides a protein feed prepared by the method described in the first aspect.

[0019] The protein feed described herein has a crude protein content of 29%–36%, of which the protein content converted from urea and inorganic ammonia nitrogen is 3%–6%, the crude fiber content is 8%–10%, the pH value is 4.2–4.8, and the live yeast count is 6–9 × 10⁻⁶. 6 CFU / g, viable Bacillus licheniformis count was 3–7 × 10⁻⁶. 6 CFU / g, viable lactic acid bacteria count is 5-6 × 10⁻⁶ 6 The CFU / g content of zearalenone is 61.2 μg / kg, and the aflatoxin content is 12.2 μg / kg. The levels of both zearalenone and aflatoxin are below the requirements of my country's "GB13078.2-2006 Feed Hygiene Standard". Unless otherwise specified, all percentages mentioned above are by weight.

[0020] Beneficial effects: Compared with the prior art, the beneficial effects achieved by the present invention are:

[0021] 1. The sprayed corn husk feed obtained by this invention uses sprayed corn husks as the main raw material, with the addition of urea. The protein feed obtained through a two-stage solid-state fermentation using Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid, and Pediococcus pentosaceus can efficiently utilize urea, achieving a urea utilization rate of 54.4%. The crude protein content increases from approximately 20% to 35%, and the true protein content increases from 10% to 20%. It can also degrade the cellulose in the sprayed corn husks, significantly increasing their nutritional value. This invention uses Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid, and Pediococcus pentosaceus, all of which are feed-grade microorganisms, for feed fermentation, which can promote digestion and absorption and ensures high safety.

[0022] 2. This invention employs a two-stage solid-state fermentation process of aerobic-anaerobic spray-fermented corn husks. Aerobic fermentation is carried out using *Aspergillus niger*, *Saccharomyces cerevisiae*, and *Bacillus licheniformis*, targeting the degradation of crude fiber and the adjustment of carbon source composition in the raw materials. Simultaneously, it degrades potential aflatoxins and zearalenones in the raw materials, allowing *Saccharomyces cerevisiae* to obtain more carbon for growth and accumulate more protein. A second-stage anaerobic fermentation is then performed using *Pediococcus pentosaceus* to address the poor palatability caused by the off-flavors introduced by *Aspergillus niger* during the first-stage aerobic fermentation. During anaerobic fermentation, the synthesized lactic acid lowers the pH of the fermented feed, inhibiting the growth of putrefactive bacteria and enhancing the flavor and palatability of the raw materials. Ultimately, this significantly improves the nutritional value of the spray-fermented corn husks, such as increasing crude protein by 80%–85%, reducing crude fiber by 20%–25%, and increasing the viable cell count to 13.16 × 10⁻⁶. 6 The CFU / g level was maintained without affecting the odor. The content of zearalenone, a toxin in the raw material, decreased from 400-450 μg / kg to 40-70 μg / kg, and the aflatoxin content was as low as 12.2 μg / kg, both below the requirements of my country's "GB13078.2-2006 Feed Hygiene Standard". This invention provides a high-protein, probiotic-rich, and palatable sprayed corn husk protein feed, which not only promotes the full and effective utilization of sprayed corn husks in the livestock industry but also effectively alleviates the shortage of feed protein resources in my country and promotes the sustainable and green development of the livestock industry. Attached Figure Description

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0024] Figure 1 pH and viable cell count changes in protein feed obtained from aerobic-anaerobic two-stage solid-state fermentation Figure 1 a represents the pH change during fermentation; Figure 1 b represents the change in the number of viable bacteria during fermentation. Figure 1 The bacteria mentioned in b are Bacillus licheniformis of the present invention, and the lactic acid bacteria mentioned are Pediococcus pentosaceus of the present invention.

[0025] Figure 2 The pH changes in the sprayed corn husk protein feed during aerobic fermentation were studied.

[0026] Figure 3 This study only investigated the changes in the viable bacterial count of the sprayed corn husk protein feed during aerobic fermentation. The bacteria mentioned are *Bacillus licheniformis* of this invention, and the lactic acid bacteria are *Pediococcus pentosaceus* of this invention. Detailed Implementation

[0027] The invention is further defined in the following embodiments. Based on the above description and these embodiments, those skilled in the art can determine the basic features of the invention, and various modifications and changes can be made to the invention without departing from its spirit and scope to make it suitable for various uses and conditions. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available. Unless otherwise specified, all techniques employed in this invention are prior art in the field.

[0028] This invention provides a two-stage solid-state fermentation method for producing protein feed from corn husks using microorganisms with urea assimilation capabilities. The *Aspergillus niger* is ATCC 16888; the *Saccharomyces cerevisiae* is ATCC 18824; the *Bacillus licheniformis* is ATCC 14580; and the *Pediococcus pentosaceus* is ATCC 33316.

[0029] In the following examples, the preparation method of the *Aspergillus niger* culture is as follows: *Aspergillus niger* is cultured on PDA solid medium at 28-32℃. After the colonies have covered the solid medium, a portion of the medium containing bacterial cells and spores is inoculated onto PDB liquid medium. The culture temperature is 28-32℃, the rotation speed is 180 rpm, and the culture time is 5-7 days. The PDA solid medium consists of the following raw materials at the following concentrations: 200 g / L potato, 20 g / L glucose, and 20 g / L agar; the PDB liquid medium consists of the following raw materials at the following concentrations: 200 g / L potato and 20 g / L glucose. The viable count of *Aspergillus niger* in the *Aspergillus niger* culture is 1.0 × 10⁻⁶. 7 CFU / g.

[0030] In the following examples, the method for preparing the *Saccharomyces cerevisiae* broth is as follows: *Saccharomyces cerevisiae* is cultured on YPD solid medium at 28-32℃. After colony formation, a single colony is picked using an inoculation loop and inoculated into YPD liquid medium for shake-flask culture at 28-32℃, 200 rpm, for 6-12 hours. The YPD solid medium consists of the following concentrations of raw materials: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, and agar 20 g / L; the YPD liquid medium consists of the following concentrations of raw materials: yeast extract 10 g / L, peptone 20 g / L, and glucose 20 g / L. The viable count of *Saccharomyces cerevisiae* in the broth is 5.0 × 10⁻⁶. 6 CFU / g.

[0031] In the following examples, the method for preparing the *Bacillus licheniformis* bacterial culture is as follows: *Bacillus licheniformis* is cultured on LB solid medium at 34-38℃. After colony formation, a single colony is picked using an inoculation loop and inoculated into LB liquid medium for shake-flask culture at 34-38℃, 220 rpm, for 6-12 hours. The LB solid medium consists of the following concentrations of raw materials: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 20 g / L agar; the LB liquid medium consists of the following concentrations of raw materials: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl. The viable count of *Bacillus licheniformis* in the bacterial culture is 0.5 × 10⁻⁶. 6 CFU / g.

[0032] In the following examples, the preparation method of the Pediococcus pentosacchari bacterial suspension (i.e., anaerobic bacterial suspension) is as follows: Pediococcus pentosacchari is cultured on MRS solid medium at 30-37℃. After colony formation, a single colony is picked up with an inoculation loop and inoculated into MRS liquid medium for shake flask culture at a temperature of 30-37℃, a rotation speed of 220 rpm, and a culture time of 6-12 hours. The MRS solid culture medium is composed of the following ingredients at the following concentrations: peptone 10 g / L, beef meal 5 g / L, yeast extract 4 g / L, glucose 20 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, diammonium citrate 2 g / L, and agar 20 g / L. The MRS liquid culture medium is composed of the following ingredients at the following concentrations: peptone 10 g / L, beef meal 5 g / L, yeast extract 4 g / L, glucose 20 g / L, Tween-80 1 mL / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, and diammonium citrate 2 g / L. The viable count of *Pediococcus pentosaceus* in the bacterial culture is 0.5 × 10⁻⁶. 6CFU / g.

[0033] Unless otherwise specified, all percentages mentioned in the following examples are mass ratios.

[0034] In the following examples, the sprayed corn husk has a crude protein content of 18%–21%, a true protein content of 8%–10%, a crude fiber content of 9%–13%, and a zearalenone content of 400–450 μg / kg.

[0035] Example 1: Two-stage solid-state fermentation method and optimization of fermentation time

[0036] After the sprayed corn husks were crushed and passed through a 40-mesh sieve, and premixed with water evenly, they were sterilized at 115–121°C. After sterilization, urea was added (3.63g of urea per 100g of dry matter, where the dry matter consists of sprayed corn husks and urea) to obtain the fermentation raw material. An aerobic bacterial solution containing Aspergillus niger, Saccharomyces cerevisiae, and Bacillus licheniformis (volume ratio 2:2:1) was inoculated into the fermentation raw material. The mass ratio of the aerobic bacterial solution to the dry matter in the fermentation raw material was 20%, and the dry matter accounted for 45% of the total fermentation mass (including the total mass of the fermentation raw material, aerobic bacterial solution, and anaerobic bacterial solution). Fermentation was carried out at 35°C for 72h, 168h, and 336h to obtain the aerobic fermentation product. Anaerobic bacterial solution was inoculated into the aerobic fermentation product at a mass ratio of 4% to dry matter, and fermented at 35°C for 24h, 48h, and 72h to obtain the protein feed. Fermentation data are shown in Table 1.

[0037] Table 1. Crude protein content in fermentation products obtained with different two-stage solid-state fermentation times.

[0038]

[0039] As shown in Table 1, the crude protein content in the anaerobic fermentation group after 72 hours of aerobic fermentation was 29%-33%, which was higher than that in the anaerobic fermentation groups after 168 hours and 336 hours of aerobic fermentation. Furthermore, the crude protein content after anaerobic fermentation decreased continuously with the extension of aerobic fermentation time, indicating that excessively long aerobic fermentation time also inhibited subsequent anaerobic fermentation. Therefore, the optimal fermentation time for the aerobic stage was 72 hours. Similarly, after 48 hours of anaerobic fermentation in each group, the increase in crude protein content decreased, and even showed a decline, indicating that the optimal time for anaerobic fermentation was 48 hours. Therefore, when anaerobic fermentation was carried out for 48 hours after 72 hours of aerobic fermentation, the crude protein content was the highest, reaching 32.30%.

[0040] Example 2: Optimization of the inoculum ratio for aerobic solid-state fermentation

[0041] After the corn husks were crushed and passed through a 40-mesh sieve, the premixed moisture was uniformly mixed and sterilized at 115-121℃. After sterilization, 3.63% urea was added (3.63g urea per 100g dry matter) to obtain fermentation raw material. Aerobic bacterial solutions (prepared in the same way as in Example 1) were inoculated at dry matter ratios of 2%, 8%, 14%, 20%, 26%, 32%, and 38% of the fermentation raw material, with the dry matter accounting for 45% of the total fermentation mass. After stirring evenly, the mixture was fermented in a 30℃ constant temperature incubator for 72 hours to obtain the aerobic fermentation product. Anaerobic bacterial solutions were inoculated into the aerobic fermentation product at a dry matter ratio of 4%, and fermented at 35℃ for 48 hours to obtain protein feed. Table 2 shows the crude protein content of the product at different inoculation amounts.

[0042] Table 2. Crude protein content in products at different inoculum sizes.

[0043]

[0044] The data in the table shows that when the ratio of aerobic bacterial inoculum to the dry weight of the fermentation raw materials is 2%-20%, the crude protein content continuously increases with the increase of this ratio, reaching its peak at 20%. When the ratio exceeds 20%, the accumulation of crude protein begins to decrease with increasing inoculum amount. Analysis suggests that insufficient inoculum leads to slow cell growth and a prolonged fermentation cycle, while excessive inoculum results in rapid cell proliferation and excessive metabolic waste. Some inoculum may undergo autolysis due to increased proteolytic enzyme activity, or secondary metabolites detrimental to cell growth may be produced during fermentation. Therefore, 20% is considered the optimal ratio of aerobic bacterial inoculum to the dry weight of the fermentation raw materials for solid-state fermentation of corn husks.

[0045] Example 3: Optimization of Aerobic Solid-State Fermentation Temperature

[0046] The preparation method of the fermentation raw materials is the same as in Example 1. An aerobic bacterial solution with a dry weight ratio of 20% to the fermentation raw materials was inoculated, and the dry matter accounted for 45% of the total fermentation mass. After stirring evenly, the mixture was placed in constant temperature incubators at 20℃, 24℃, 28℃, 32℃, 36℃, 40℃, and 44℃ for 72 hours to obtain aerobic fermentation products. Anaerobic bacterial solution was then inoculated into the aerobic fermentation products at a dry matter ratio of 4%, and fermented at 35℃ for 48 hours to obtain protein feed. Table 3 shows the crude protein content of the products at different culture temperatures.

[0047] Table 3 Crude protein content at different fermentation temperatures

[0048]

[0049] When the fermentation temperature is within the range of 20-36℃, the crude protein content of the fermentation product gradually increases with increasing temperature. It reaches its highest value at 36℃, with a crude protein content of 34.33%, which is 21.3% higher than the crude protein content at 0 hours of fermentation (28.3%). Above 36℃, the crude protein content gradually decreases with increasing temperature. Fermentation temperature affects the structure and function of proteins and other molecules, thus influencing the growth, proliferation, and metabolic activities of the fermenting bacteria. Different fermentation temperatures result in different nutrient contents in the fermentation products. This may be because at too low a temperature, the bacterial cell division and proliferation are too slow, inhibiting the activity of some secreted enzymes and cellulase, thus hindering their enzymatic hydrolysis and affecting the feed fermentation effect. At too high a temperature, the activity of some enzymes decreases, affecting bacterial growth and the enzymatic hydrolysis of fermentation quality, resulting in poor fermentation. Therefore, 36℃ is the optimal temperature for solid-state fermentation of spray-dried corn husks.

[0050] Example 4: Optimization of the dry matter ratio in fermentation feedstock

[0051] After the corn husks were crushed and passed through a 40-mesh sieve, the premixed moisture was uniformly mixed and sterilized at 115-121℃. After sterilization, 3.63% urea was added (same as in the above example), and aerobic bacterial solution with a dry weight ratio of 20% to the fermentation raw materials was inoculated. The dry matter as a percentage of the total fermentation mass was 15%, 25%, 35%, 45%, 55%, 65%, and 75%, respectively. After stirring evenly, the mixture was placed in a 36℃ constant temperature incubator for 72 hours to obtain the aerobic fermentation product. Anaerobic bacterial solution was inoculated into the aerobic fermentation product at a dry matter mass ratio of 4%, and fermented at 35℃ for 48 hours to obtain the protein feed. Table 4 shows the crude protein content of the product at different moisture contents.

[0052] Table 4 Crude protein content at different dry matter ratios

[0053]

[0054] When the dry matter percentage is 35%, the crude protein content is highest at 34.97%. Below 35%, the crude protein content gradually decreases, and above 35%, it decreases significantly. The dry matter percentage has a significant impact on the crude protein content of fermented feed products. At 25%, the fermentation feed is semi-liquid, hindering air circulation. A dry matter percentage between 35% and 45% is suitable, resulting in relatively loose feed particles. Between 45% and 75%, the feed particles clump together, potentially affecting microbial growth, proliferation, and metabolism. Based on this analysis, a dry matter percentage of 35% indicates optimal looseness of the fermentation feed, with some porosity between particles, facilitating water and air circulation and ensuring normal dissolved oxygen transport. Therefore, a dry matter percentage of 35% yields the best fermentation effect, resulting in a maximum crude protein content of 34.97%, approximately 23.6% higher than the crude protein content at 0 hours of fermentation (28.3%).

[0055] Example 5: Production of Sprayed Corn Husk Protein Feed via Two-Stage Solid-State Fermentation under Optimal Fermentation Conditions

[0056] After the corn husks were crushed and passed through a 40-mesh sieve, the mixture was premixed with water and sterilized at 115-121℃. After sterilization, 3.63% urea was added, and an aerobic bacterial solution with a dry weight ratio of 20% to the fermentation raw material was inoculated. The dry matter accounted for 35% of the total fermentation mass. After thorough mixing, the mixture was placed in a 36℃ constant temperature incubator for 72 hours of fermentation. Then, the fermented feed was transferred to an anaerobic fermentation bag and inoculated with lactic acid bacteria (anaerobic bacterial solution) for anaerobic fermentation at 36℃ for 48 hours. Table 5 shows the nutritional indicators of the corn husk protein feed produced by two-stage solid-state fermentation under optimal fermentation conditions.

[0057] Table 5. Nutritional composition analysis of feed products during two-stage solid-state fermentation.

[0058]

[0059] Note: 1 kg of urea contains approximately the same amount of nitrogen as 2.91 kg of protein. Urea-to-protein ratio = urea mass × 2.91

[0060] 3.63g of urea was added to 100g of spray-fermented corn husks after 0 hours of fermentation. After 72 hours of aerobic fermentation and 48 hours of anaerobic fermentation, the crude protein content continued to increase to 35%, which is 80%–85% higher than that of spray-fermented corn husks; the true protein content increased to 20.3% (true protein content: the protein in the feed product is precipitated by the hot alkaline method, then filtered through filter paper, and the precipitate and filter paper are determined by Kjeldahl nitrogen determination), which is 2–2.5 times that of spray-fermented corn husks; the protein content converted from urea nitrogen and ammonia nitrogen was 5.75%; the crude fiber decreased to 9.9%; and the urea utilization rate was 54.4%. Through aerobic-anaerobic two-stage solid-state fermentation, the pH of the feed product was maintained below 5. Figure 1 a) The viable yeast count is 6–9 × 10⁻⁶. 6 CFU / g, viable Bacillus licheniformis count was 3–7 × 10⁻⁶. 6 CFU / g, viable lactic acid bacteria count is 5-6 × 10⁻⁶ 6 CFU / g ( Figure 1 b). Furthermore, the protein feed did not have a noticeable off-odor.

[0061] Through two-stage solid-state fermentation, the total dry weight loss rate of the fermented material is less than 30%, and the urea conversion rate reaches 54%. This indicates that combining multiple microorganisms with urea utilization capabilities for two-stage solid-state fermentation can effectively improve the efficiency of urea conversion into protein, reduce material loss during fermentation, and lower the cost issues caused by material loss during production.

[0062] Zearalenone and aflatoxin are two toxins that are most likely to exceed the limits in spray-dried corn husks. Therefore, the contents of zearalenone and aflatoxin in corn before and after aerobic-anaerobic fermentation were measured. The zearalenone content was reduced to 61.2 μg / kg, which improved the quality of the spray-dried corn husks after fermentation. The aflatoxin content was 12.2 μg / kg, which meets the national feed safety standards.

[0063] Comparative Example 1

[0064] After the corn husks were crushed by spraying, they were passed through a 40-mesh sieve. After premixing the moisture evenly, the mixture was sterilized at 115-121℃. After sterilization, 3.63% urea was added. Microorganisms (Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid and Pediococcus pentosaceus liquid in a volume ratio of 2:2:1:1) were inoculated with a dry weight ratio of 24% to the fermentation raw materials. The solid fermentation dry matter accounted for 35%. After stirring evenly, the mixture was incubated in a 30℃ constant temperature incubator for aerobic fermentation for 336 hours. The crude protein content, pH and viable cell count were measured at 0h, 72h, 168h and 336h, as shown in Table 6.

[0065] Table 6. Changes in products at different fermentation times

[0066]

[0067] Using crude protein content as an indicator, the highest crude protein content of 32.3% was achieved after 72 hours of fermentation.

[0068] At the same time, the change in pH value during fermentation was measured. Figure 2 It was found that the feed, initially at pH 4.53, increased to pH 5.2 after 72 hours of aerobic fermentation, and its odor worsened. After 72 hours of fermentation, the pH gradually rose to neutral, which was detrimental to feed preservation. Furthermore, the protein feed obtained using this fermentation method had a viable bacterial count of only 2.47 × 10⁻⁶. 6 The CFU / g of fermented materials was low, with a urea utilization rate of only 28.8%. The total dry weight loss of the fermented materials reached 40%. The zearalenone content was 304.1 μg / kg, and the aflatoxin content was 16.7 μg / kg.

[0069] This invention provides a method for producing protein feed from corn husks through a two-stage solid-state fermentation spraying process. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for producing protein feed from corn husks through two-stage solid-state fermentation using microorganisms, characterized in that, Includes the following steps: Aerobic bacterial solution is inoculated into fermentation raw materials for aerobic solid-state fermentation to obtain aerobic fermentation products; anaerobic bacterial solution is inoculated into aerobic fermentation products for anaerobic solid-state fermentation to obtain protein feed. The aerobic bacterial solution includes Aspergillus niger solution, Saccharomyces cerevisiae solution, and Bacillus licheniformis solution; The anaerobic bacterial solution includes Pediococcus pentosaceus bacterial solution; The fermentation raw materials include dry matter, which is sprayed corn husks and urea, wherein the mass ratio of urea in the dry matter is 1% to 5%. The aforementioned aerobic solid-state fermentation is carried out at a temperature of 22~44℃ for a fermentation time of 72~168 h. The anaerobic solid-state fermentation is carried out at a temperature of 20-40 °C for 24-72 h. The viable count of Aspergillus niger in the Aspergillus niger solution is 0.5~2×10⁻⁶. 7 CFU / g; the viable count of brewer's yeast in the brewer's yeast culture is 4~6×10⁻⁶. 6 CFU / g; the viable count of Bacillus licheniformis in the bacterial culture is 0.1~1.0×10⁻⁶. 6 CFU / g; the viable count of *Pediococcus pentosaceus* in the bacterial culture was 0.1~1.0×10⁻⁶ CFU / g. 6 CFU / g; The volume ratio of the Aspergillus niger liquid, Saccharomyces cerevisiae liquid, Bacillus licheniformis liquid, and Pediococcus pentosaceus liquid is 1~2: 1~2: 0.5~1: 0.5~1.

2. The method according to claim 1, characterized in that, The aforementioned Aspergillus niger is Aspergillus niger (Aspergillus niger) Aspergillus niger ATCC 16888; the brewing yeast mentioned is brewing yeast ( Saccharomyces cerevisiae ATCC18824; The Bacillus licheniformis mentioned is Bacillus licheniformis ( Bacillus licheniformis ATCC 14580; the Pediococcus pentosaceus mentioned is Pediococcus pentosaceus ( Pediococcus pentosaceus ATCC 33316.

3. The method according to claim 1, characterized in that, The dry matter accounts for 10% to 80% of the total fermentation mass.

4. The method according to claim 3, characterized in that, The mass ratio of the aerobic bacterial solution to dry matter is 1% to 50%.

5. The protein feed prepared by the method according to any one of claims 1 to 4, wherein the protein feed has a crude protein content of 29% to 36%, wherein the protein content converted from urea and inorganic ammonia nitrogen is 3% to 6%, the crude fiber content is 8% to 10%, the pH value is 4.2 to 4.8, and the viable yeast count is 6 to 9 × 10⁻⁶. 6 CFU / g, viable Bacillus licheniformis count is 3~7×10⁻⁶. 6 CFU / g, viable lactic acid bacteria count is 5~6×10⁻⁶ 6 The CFU / g content of zearalenone was 61.2 μg / kg, and the aflatoxin content was 12.2 μg / kg.

6. The use of the protein feed according to claim 5 in the preparation of animal feed.

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