Compound feed additive and preparation method thereof
Through the co-fermentation of Aspergillus niger and Max Kluvieriaces, the feedback inhibition problem of enzyme production in deep fermentation of liquid is solved, the nutritional digestibility of feed and animal health is improved, the production process is simplified, and the safety of genetically engineered bacteria is avoided. Sexual complexity.
Patent Information
- Application Number
- CN202411283852.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-13
- Publication Date
- 2024-12-31
AI Technical Summary
Prior Art During the deep fermentation of liquids, xylanases and acid proteases produced by Aspergillus niger are inhibited by feedback from catabolites such as amino acids, which leads to difficulties in full use of enzymes, and safety approval and production license of genetically engineered bacterial strains. The problems are complex and affect product quality and production efficiency.
By co-fermenting Aspergillus niger and Max Kluvieriaces, by optimizing the fermentation medium and selecting the appropriate bacterial strain combination, Max Kluvieriaces eliminates feedback inhibitors, and improves the production efficiency of Aspergillus niger's xylanase and acid proteases. , form composite feed additives.
It significantly improves the digestion and absorption rate of nutrients such as protein in the feed, reduces the amount of protein raw materials, improves the health of animals, avoids the safety of genetically engineered bacteria, and simplifies the production process.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a compound feed additive produced by liquid deep fermentation and a preparation method thereof. Background Art
[0002] The shortage of feed resources has always been a bottleneck problem restricting the development of my country's feed industry, especially the shortage of protein feed resources. In my country, the main sources of protein feed resources are plant raw materials such as soybean meal and other plant meal such as rapeseed meal, cotton meal and peanut meal.
[0003] Hemicellulose is the main component of plant cell walls, and xylan is the main chemical component of plant hemicellulose, which is sticky and indigestible to the intestines of monogastric animals. Xylanase can decompose the hemicellulose of plant cell walls, promote the release of effective substances by destroying the "cage effect" of xylan, reduce the viscosity of chyme, and increase the activity of endogenous digestive enzymes. It plays a beneficial role in the intestines of animals and promotes the digestion and absorption of nutrients. Studies have found that after adding xylanase to corn-soybean meal diets, dietary components such as starch, crude protein, amino acids, and various minerals in the intestines of pigs have increased to varying degrees, and the digestibility of intestinal nutrients has also increased in a nonlinear manner with the change of xylanase concentration.
[0004] Acidic proteases are widely used in the feed industry. Large-scale livestock, poultry and aquatic products, especially young animals in breeding, have poor digestion of feed protein due to insufficient enzyme system and enzyme quantity (endogenous enzyme) secreted by their digestive organs, which can easily cause indigestion, diarrhea and other diseases. Studies have found that adding acidic proteases to feed can help improve the digestibility of protein and degrade protein into amino acids and polypeptides that are easy for young animals to digest and absorb, thereby reducing the use of plant proteins such as plant meal, and can also reduce nitrogen emissions in feces in the breeding industry, reduce environmental pollution, and reduce the pressure on environmental protection measures.
[0005] Aspergillus niger is a common strain in filamentous fungi, which can produce a variety of enzymes such as amylase, protease, xylanase, cellulase, pectinase, etc. The xylanase and acid protease produced by Aspergillus niger are induced enzymes, that is, they can be secreted in large quantities in the presence of substrates such as xylan and macromolecular proteins. At present, in the fermentation production of feed enzyme preparations, some processes use Aspergillus niger to produce xylanase, cellulase and protease by single strain fermentation, but in this method, Aspergillus niger produces amino acid and other decomposition metabolites while producing enzymes, especially in the process of deep liquid fermentation, the xylose, amino acid and other decomposition metabolites produced by Aspergillus niger fermentation produce feedback inhibition on the further secretion of xylanase and protease, so that the enzyme production level of Aspergillus niger cannot be fully exerted. In addition, some processes use transgenic technology to transform Aspergillus niger into strains, so as to obtain transgenic feed microbial strains. However, in today's world where product safety is emphasized, there are many problems in the fermentation process using genetically engineered bacteria, including safety approval and production license of genetically modified bacteria, separation of bacteria and enzymes, and post-treatment of genetically modified bacteria after fermentation production. It takes a long time and cost to ensure the smooth progress of the fermentation production process of genetically engineered bacteria, quality assurance and licensing of products, while the non-genetically engineered bacteria Aspergillus niger and Kluyveromyces marxianus used in the present invention do not have such problems.
[0006] Therefore, in view of the current shortage of feed resources in my country, there is an urgent need to produce a feed additive that can promote the digestion and absorption of nutrients such as protein in feed by livestock and poultry, reduce the use of protein raw materials such as soybean meal, and improve the health of animals. Summary of the invention
[0007] In view of the problems existing in the prior art, the present invention provides a composite feed additive produced by liquid deep fermentation and a preparation method thereof, wherein the fermented product includes xylanase and acid protease, which are obtained by co-fermentation of Aspergillus niger and Kluyveromyces marxianus with soybean meal and defatted rice bran as fermentation substrates. The feed additive provided by the present invention can promote the digestion and absorption of nutrients such as protein in feed by livestock and poultry, and improve the health of animals.
[0008] On the one hand, the present invention provides a fermentation product, which is obtained by pre-fermentation of Aspergillus niger and then adding Kluyveromyces marxianus for co-fermentation.
[0009] In order to solve the feedback inhibition problem of Aspergillus niger in the process of producing xylanase and acid protease in liquid deep fermentation, the present invention considers adding other strains for co-fermentation and optimizing the fermentation medium, hoping to better exert the fermentation effect of Aspergillus niger.
[0010] By using Aspergillus niger and Kluyveromyces marxianus, Candida utilis, Bacillus subtilis and Lactobacillus plantarum for combined fermentation screening, the results showed that since Bacillus requires high temperature conditions during the fermentation process, and lactic acid bacteria fermentation requires an anaerobic environment, the two species cannot be fermented at the same time with Aspergillus niger. Therefore, the enzyme production levels of the two combined fermentation results of Aspergillus niger and Bacillus subtilis and Lactobacillus plantarum were lower than those of the fermentation results using Aspergillus niger alone. The fermentation results of the combination of Aspergillus niger and Candida utilis were lower than those of the combination of Aspergillus niger and Kluyveromyces marxianus. Therefore, the combination of Aspergillus niger and Kluyveromyces marxianus was finally selected for fermentation.
[0011] The yeast used in the present invention must be a yeast that can utilize xylose, such as Kluyveromyces marxianus, Candida utilis, etc., among which Kluyveromyces marxianus is the most preferred.
[0012] Aspergillus niger is an important industrial fermentation strain that can produce important enzymes such as amylase, protease, xylanase, cellulase, pectinase, etc. The metabolites of Aspergillus niger can improve the nutritional level and digestibility of animal feeds, and can decompose large molecular proteins in feeds into small molecular amino acids and starch into monosaccharides. Non-starch polysaccharide enzymes such as xylanase produced by metabolism eliminate anti-nutritional factors in feeds, greatly improve the nutritional level and digestibility of feeds, and thus effectively promote the health, growth and weight gain of animals.
[0013] Kluyveromyces marxianus is a commonly used microecological preparation strain. The fermentation culture of this strain contains yeast cells and extracellular metabolites and autolysis products such as peptides, organic acids, oligosaccharides, amino acids, flavor enhancers and aromatic substances. Compared with brewer's yeast, this yeast has an obvious advantage in that it can effectively utilize xylose, the enzymatic hydrolysis product of xylan, to convert it into its own protein. Xylose is a pentose that is difficult for monogastric animals to absorb and utilize. Decades of scientific research and production application practice have proved that yeast fermentation culture can effectively regulate the number and proportion of microorganisms in the animal intestine, inhibit the survival of pathogens, thereby improving the balance of its intestinal flora, and can significantly reduce the number of harmful microorganisms and diarrhea rate in the animal intestine, improve the health of animals, and thus significantly improve the productivity of farmed animals.
[0014] Furthermore, the fermentation product comprises xylanase and acid protease.
[0015] Xylanase can degrade xylan to destroy plant cell walls, eliminate anti-nutritional factors, and improve feed utilization. At the same time, it can reduce the viscosity of digestive tract contents, improve animal production performance, and improve the intestinal flora structure of animals, reduce harmful bacteria, thereby increasing animal feed intake and daily weight gain, and reducing feed-to-meat ratio. Acidic proteases are mainly used to supplement the deficiency of endogenous proteases in animals, especially to promote the digestion and absorption of protein by young animals, improve the digestibility of high-protein diets in growing animals, and reduce the amount of protein raw materials and formulation costs. Acidic proteases can reduce the fermentation of harmful microorganisms in the hindgut, reduce the incidence of nutritional diarrhea, promote animal intestinal health, increase daily weight gain, reduce feed-to-weight ratio, and improve animal production performance.
[0016] Furthermore, the fermented product uses soybean meal and defatted rice bran as fermentation substrates.
[0017] The formula of the fermentation medium will directly affect the growth of the strain and the final product generation. The present invention screened the components of the main fermentation tank medium and found that when soybean meal and defatted rice bran are used as fermentation medium substrates, the level of xylanase and acid protease produced by Aspergillus niger can be significantly improved.
[0018] In some embodiments, different species of Aspergillus niger can be used for submerged fermentation.
[0019] In another aspect, the present invention provides a compound feed additive, comprising the fermentation product as described above.
[0020] In another aspect, the present invention provides a method for producing a compound feed additive by using liquid submerged fermentation, wherein the compound feed additive is prepared by mixed fermentation of Aspergillus niger and Kluyveromyces marxianus.
[0021] Furthermore, the method comprises the following steps:
[0022] (1) Seed culture of Aspergillus niger and Kluyveromyces marxianus;
[0023] (2) Early fermentation culture of Aspergillus niger;
[0024] (3) Mixed fermentation culture of Aspergillus niger and Kluyveromyces marxianus;
[0025] (4) Post-treatment of fermentation liquid.
[0026] Furthermore, in step (1), the seed culture medium uses soybean meal as an organic nitrogen source and corn flour as a carbon source.
[0027] Furthermore, the culture medium for the preliminary fermentation in step (2) and the mixed fermentation in step (3) contains soybean meal as an organic nitrogen source and defatted rice bran as a carbon source.
[0028] On the other hand, the present invention provides a use of a fermentation product for preparing a preparation for improving the digestibility of nutrients such as protein in feed raw materials, wherein the fermentation product is obtained by co-fermentation of Aspergillus niger and Kluyveromyces marxianus through liquid submerged fermentation.
[0029] In another aspect, the present invention provides a use of a yeast for preparing a preparation for eliminating feedback inhibition factors produced during the fermentation process of Aspergillus niger, characterized in that the yeast is Kluyveromyces marxianus.
[0030] Aspergillus niger produces xylanases, acid proteases and other enzymes under the induction of fermentation substrates, and at the same time produces xylose and amino acids. The xylose and amino acids produced by Aspergillus niger fermentation have a feedback inhibitory effect on the further secretion of xylanase and protease.
[0031] The invention uses Kluyveromyces marxianus, which can better utilize xylose and amino acids, and Aspergillus niger for mixed fermentation, effectively eliminates feedback inhibition factors, promotes Aspergillus niger fermentation to produce xylanase and acid protease, improves fermentation enzyme activity, and simultaneously produces Kluyveromyces marxianus, a microecological preparation.
[0032] Compared with the prior art, the present invention has the following advantages:
[0033] 1. Kluyveromyces marxianus can make good use of xylose and amino acids produced by Aspergillus niger fermentation, effectively eliminate feedback inhibition factors, and promote the production of xylanase and acid protease by Aspergillus niger fermentation;
[0034] 2. The culture medium components and the mixed fermentation method of selecting the inoculation sequence provided by the present invention can effectively improve the activity of xylanase and acidic protein;
[0035] 3. The feed additive provided by the present invention can promote the digestion and absorption of nutrients by livestock and poultry and improve the health of animals. DETAILED DESCRIPTION
[0036] The Kluyveromyces marxianus strain with the accession number CICC1218 and the Aspergillus niger strain with the accession number CCTCC NO: M202018 involved in the present invention are not introduced in detail because they do not belong to the innovative content of the present invention.
[0037] The present invention is further described in detail below in conjunction with the examples. It should be noted that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. The reagents used in this example are all known products and were obtained by purchasing commercially available products.
[0038] Example 1: Preparation of the compound feed additive provided by the present invention
[0039] (1) Cultivation of Aspergillus niger (strain collection number: CCTCC NO: M202018) seeds
[0040] The slant-preserved strains of Aspergillus niger were inoculated into the triangular flask culture medium and then cultured on a shaking table at 30°C and 200rpm for 18 to 20h; then the triangular flask seeds were inoculated into the primary seed tank for 18 to 20h, and the culture conditions were: 30°C, 180 to 200rpm, and the ventilation volume was 1:0.5 to 0.6 (V / V); the seeds cultured in the primary seed tank were inoculated into the secondary seed tank at a 5% inoculation rate, and the culture conditions were: 30°C, 160 to 180rpm, and the ventilation volume was 1:0.5 to 0.6 (V / V), and cultured for 16 to 18h, and then used. The formula of the culture medium for triangular flask seeds, primary seed tanks, and secondary seed tanks is as follows:
[0041] Soybean meal powder 30g / L, corn powder 10g / L, ammonium chloride 4g / L, dipotassium hydrogen phosphate 2g / L. Natural pH, sterilized at 121℃ for 30min.
[0042] (2) Slant seed culture of Kluyveromyces marxianus (strain collection number CICC 1218) in a flask
[0043] The strains of Kluyveromyces marxianus preserved on the slant surface of the flask were inoculated and cultured at 30°C for 48 hours to obtain Kluyveromyces marxianus seeds. The slant surface culture medium of the flask was solid PDA, and the specification of the flask was 250 mL.
[0044] (3) Cultivation of the early fermentation of Aspergillus niger
[0045] The seeds from the seed tank obtained in step (1) are inoculated into the main fermentation tank at a 5% inoculation rate, and fermented and cultured at 30°C, 160-180rpm, and a ventilation rate of 1:0.5-0.6 (V / V) for 18-20 hours for standby use. The main fermentation tank culture medium formula is as follows:
[0046] Soybean meal 50g / L, defatted rice bran 25g / L, ammonium chloride 4g / L, calcium chloride 5g / L, potassium dihydrogen phosphate 8g / L. Natural pH, sterilized at 121℃ for 30min.
[0047] (4) Mixed fermentation of Aspergillus niger and Kluyveromyces marxianus
[0048] The Kluyveromyces marxianus seeds cultured in step (2) are inoculated in an amount of one flask slope per 1000L of fermentation liquid, and the eluted yeast seeds are inoculated into the fermentation liquid of the main fermentation tank in step (3), and the culture is continued for 58 to 60 hours at 30°C, 160 to 180 rpm, and a ventilation volume of 1:0.6 to 0.7 (V / V).
[0049] (5) Post-treatment of fermentation liquid:
[0050] The fermentation liquid obtained in step (4) can be directly adsorbed with an adsorbent to form a solid preparation product; or a precipitant can be added and then subjected to solid-liquid separation to separate and remove the bacteria from the fermentation liquid, and then made into a sterile liquid enzyme preparation product or adsorbed to form a solid preparation product according to product requirements.
[0051] Example 2: Screening of different strains
[0052] Screening fermentation strains is the key to microbial fermentation and enzyme production. Generally speaking, first, the strains must comply with safety principles and cannot produce toxic and harmful substances. Second, the strains have good growth and metabolic activity, can grow rapidly in the culture medium, and effectively secrete the required enzymes and other metabolites. Lactic acid bacteria, yeast, Bacillus, etc. are the most common feeding microbial strains. This embodiment provides fermentation strains with different combinations of Aspergillus niger for preparing compound feed additives. The remaining processes and steps are the same as in Example 1, and the control is to inoculate Aspergillus niger only to screen the best strain combination. The xylanase activity was determined by the DNS method, and the acid protease activity was determined by the Folin method. The measurement results are shown in Table 1.
[0053] Table 1 Determination of xylan and acid protease activity under different bacterial combinations
[0054]
[0055] Combinations 1 to 4 are compared with the control, and it can be seen that the xylanase activity is 22500U / mL and the acid protease activity is 4140U / mL in the strain combination (combination 3) provided by the present invention, which significantly improves the production efficiency of xylanase and acid protease compared to the control; Aspergillus niger and Bacillus subtilis co-fermentation (combination 1), the xylanase activity is 6400U / mL, and the acid protease activity is 1020U / mL; Aspergillus niger and Lactobacillus plantarum co-fermentation (combination 2), the xylanase activity is 10200U / mL, and the acid protease activity is 1710U / mL. Since Bacillus requires high temperature conditions during the fermentation process, and lactic acid bacteria fermentation requires an anaerobic environment, the simultaneous fermentation of the two strains with Aspergillus niger is not ideal, and the enzyme activity is reduced compared with the control. When Candida utilis replaced Kluyveromyces marxianus (combination 4), the xylanase activity was 17900U / mL and the acid protease activity was 3760U / mL. Although Candida utilis and Kluyveromyces marxianus can both utilize xylanase, there is a significant difference in the effect of combined fermentation with Aspergillus niger. According to the above results, the compound feed additive prepared with Kluyveromyces marxianus has the best effect.
[0056] Example 3: Screening of bacterial combination fermentation process
[0057] In production practice, it is often necessary to select suitable fermentation strains and processes in combination with factors such as animal needs and fermentation substrates. The fermentation methods and steps of different strain combinations will also affect the final fermentation products. Therefore, in order to further explore the effect of the fermentation process on the final two enzyme activities, this example provides a fermentation method as shown in Table 2, and other conditions are the same as in Example 1.
[0058] Table 2 Effects of different fermentation methods on the activity of xylanase and acid protease
[0059]
[0060] As can be seen from Table 2, when inoculating Aspergillus niger and Kluyveromyces marxianus (mode 1) at the same time, the xylanase activity is 8400U / mL, and the acid protease activity is 1080U / mL, because Aspergillus niger and Kluyveromyces marxianus ferment at the same time, the growth of Kluyveromyces marxianus and the growth of Aspergillus niger form a competitive relationship, which greatly affects the growth and reproduction of Aspergillus niger mycelium, causing the production efficiency of Aspergillus niger enzyme production to decrease; and when Aspergillus niger is first cultivated for a period of time under the conditions of the main fermentation tank culture medium, Aspergillus niger has fully grown, and the xylose and amino acids produced reach a certain level, and now adding Kluyveromyces marxianus co-fermentation will not affect the growth and reproduction of the thalli of Aspergillus niger, and now yeast can utilize the decomposition metabolites of Aspergillus niger to carry out limited growth and reproduction, reducing the feedback inhibition factor of Aspergillus niger enzyme production, and effectively improving the production efficiency of xylanase and acid protease. Therefore, the preferred fermentation mode 2 of the present embodiment is fermented.
[0061] Example 4: Screening of culture medium formulas for triangular flask seeds, primary seed tanks, and secondary seed tanks
[0062] Fermentation medium is used for bacterial growth, reproduction and product synthesis. It should not only enable the seeds to grow rapidly after inoculation and reach a certain mycelium concentration, but also enable the grown bacteria to quickly synthesize the required products. Therefore, the composition of the fermentation medium should be rich and complete. The components of microbial fermentation medium mainly include carbon source, nitrogen source, inorganic salts and trace elements, growth factors, water, as well as inducers, precursors and promoters. Common nitrogen sources include soybean cake (meal) powder, peanut cake powder, fish meal, ammonium salts, etc., and inorganic salts include potassium salts, trace elements, etc.
[0063] In order to further explore the effect of the composition of the seed tank culture medium on the activity of the two enzymes produced in the main fermentation tank, this example provides different seed tank culture mediums, the composition of which is shown in Table 3, and the remaining processes and steps are the same as those in Example 1. The activity determination results of xylanase and acid protease are shown in Table 3.
[0064] Table 3 Effects of different seed tank culture medium components on xylanase and acid protease activities
[0065]
[0066] According to Table 3, by comparing formulas 1 to 4, it can be seen that the xylanase activity in formula 1 is 22600, and the acid protease activity is 4080. When the soybean meal and corn flour components in the culture medium are replaced with other substances, the xylanase activity decreases, and the acid protease activity decreases; when the ammonium chloride component is replaced with calcium chloride, the xylanase activity is 20600, the acid protease activity is 3470, and the enzyme production efficiency decreases. The above results show that the compound feed additive prepared by formula 1 has the best effect.
[0067] Example 5: Screening of culture medium formulation for main fermentation tank
[0068] In order to explore whether the main fermentation tank culture medium components have an effect on the activities of the two enzymes, this example provides four main fermentation tank culture media as shown in Table 4, and the remaining processes and steps are the same as those in Example 1. The activity determination results of xylanase and acid protease are shown in Table 4.
[0069] Table 4 Effects of different main fermentation tank culture medium components (substance content remains unchanged) on the activity of xylanase and acid protease
[0070]
[0071] According to Table 4, it can be seen from the comparison of formulas 5 to 8 that when the soybean meal and defatted rice bran in the culture medium are replaced with other substances, the xylanase activity and acid protease activity are reduced; when the potassium dihydrogen phosphate component is replaced with sodium dihydrogen phosphate, the xylanase activity is 21600U / mL, the acid protease activity is 3620U / mL, and the enzyme production efficiency is reduced. It can be seen that the compound feed additive prepared by formula 5 has the best effect, so formula 5 is preferred in this embodiment.
[0072] Example 6: Feeding test of the compound feed additive provided by the present invention for weaned piglets
[0073] In this embodiment, 88 28-day-old "Du Dachang" weaned piglets (half male and half female) were selected, all piglets were weighed individually, and divided into a control group and a test group according to weight and gender, each treatment had 4 replicates, and each replicate had 11 piglets. The control group was fed a basic diet, and the basic diet formula was:
[0074] Corn 62%, soybean meal 13.8%, puffed soybeans 10.0%, fish meal (Peru) 5.0%, whey powder 6.0%, calcium hydrogen phosphate 1.2%, stone powder 0.6%, salt 0.3%, lysine 0.1%, premix 1.0%.
[0075] The experimental group's diet was based on the basic diet, with 2 kg of bran powder adsorbed from the fermentation broth of Example 1 added per ton of feed (2 kg was directly adsorbed from 2 L of Aspergillus niger fermentation broth, i.e., the feed additive in Example 1).
[0076] The experimental period was 28 days. The piglets were weighed on the 0th and 28th days of the experiment, and the feed consumption was recorded. The average daily growth, average daily feed intake and feed-to-weight ratio of the piglets were calculated. The results are shown in Table 5.
[0077] Table 5 Results of piglet production performance test in different treatment groups
[0078]
[0079] Note: aa indicates a significant increase compared with the control group, p<0.05; b indicates a significant decrease compared with the control group, p<0.05.
[0080] As can be seen from Table 5, after the compound feed additive produced in Example 1 was added to the feed, the average daily feed intake of piglets was significantly increased (p<0.05), and the feed-to-weight ratio was significantly reduced (p<0.05). The above animal test results show that the addition of xylanase and acid protease produced by Aspergillus niger and yeast complex in Example 1 to the feed can significantly improve the production performance of weaned piglets.
[0081] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.
Claims
1. A fermented product produced by submerged fermentation, characterized in that: The fermented product is firstly fermented by Aspergillus niger, and then Kluyveromyces marxianus is added for co-fermentation to obtain the product.
2. The fermentation product according to claim 1, characterized in that Includes xylanase and acid protease.
3. The fermentation product according to claim 2, characterized in that Soybean meal and defatted rice bran were used as fermentation substrates.
4. A compound feed additive, characterized in that: The method comprises the fermentation product according to any one of claims 1 to 3.
5. A method for producing a compound feed additive by using liquid submerged fermentation, characterized in that: It is prepared by mixed fermentation of Aspergillus niger and Kluyveromyces marxianus.
6. The method according to claim 5, characterized in that The following steps are involved: (1) Seed culture of Aspergillus niger and Kluyveromyces marxianus; (2) Early fermentation culture of Aspergillus niger; (3) Mixed fermentation culture of Aspergillus niger and Kluyveromyces marxianus; (4) Post-treatment of fermentation liquid.
7. The method according to claim 6, characterized in that The culture medium for seed culture in step (1) contains soybean meal as an organic nitrogen source and corn meal as a carbon source.
8. The method according to claim 7, characterized in that The culture medium for the preliminary fermentation in step (2) and the mixed fermentation in step (3) contains soybean meal as an organic nitrogen source and defatted rice bran as a carbon source.
9. Use of a fermented product for preparing a preparation for improving the digestibility of protein in feed, characterized in that: The fermented product is obtained by co-fermentation of Aspergillus niger and Kluyveromyces marxianus.
10. A use of yeast for preparing a preparation for eliminating feedback inhibition factors produced during Aspergillus niger fermentation, characterized in that: The yeast is Kluyveromyces marxianus.