Fermented feed prepared using Ganoderma lucidum, and preparation method and application thereof

Abstract

The present application belongs to the technical field of fermented feed, and particularly relates to a fermented feed prepared by using ganoderma lucidum, and a preparation method and application thereof. The present application takes by-products such as vinasse, cassava residue, potato residue, kelp residue, pepper residue and traditional Chinese medicine residue as substrates, through screening of fermentation strains, optimization of fermentation process and fermentation conditions, and by using liquid fermentation technology, a functional feed with low fermentation cost and rich in nutritional components is prepared by expanding the use of the above-mentioned unconventional feed resources, so that the purpose of replacing soybean meal as animal feed can be achieved, and food is saved. Meanwhile, the present application uses ganoderma lucidum as a fermentation strain, and the product after fermentation contains ganoderma polysaccharide, which can effectively protect the intestinal tract of animals and maintain the health of the intestinal tract of animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented feed, and in particular relates to a fermented feed and a preparation method and application thereof. Background Art

[0002] In recent years, feed resources have been in short supply. In particular, concentrated feed resources (such as corn, soybean meal, fish meal, etc.) are in short supply and are relatively expensive, while cheap roughage is discarded or burned in large quantities because it cannot be fully utilized by animals, resulting in resource waste and environmental pollution. To this end, agricultural by-products can be fully utilized to make up for the shortage of high-quality protein. The latest "Feed Raw Materials Catalog" includes 43 kinds of by-products such as dregs after the extraction of plant raw materials, by-products of food and fruit and vegetable production processes, and 117 kinds of traditional Chinese medicine raw materials. These raw materials are collectively referred to as biomass resources. As a traditional agricultural powerhouse, my country has a rich variety of biomass and a huge total amount. The large raw material base provides good conditions for the development and utilization of biomass feed.

[0003] Based on the above situation, this study uses distiller's grains, cassava residue, potato residue, kelp residue, chili residue and traditional Chinese medicine residue as raw materials, screens fermentation strains, optimizes fermentation processes, and utilizes liquid fermentation technology to prepare feed, thereby achieving the goal of partially replacing soybean meal as high-quality protein feed for animals. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the existing technology and to provide a fermented feed by utilizing biotechnology to process unconventional feed and expand the utilization of unconventional feed resources.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the fermented feed.

[0006] The final technical problem to be solved by the present invention is to provide an application of the fermented feed.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A fermented feed prepared using Ganoderma lucidum, wherein the fermented feed is prepared by fermenting fermentation raw materials;

[0009] Wherein, the fermentation raw materials account for 60-80% of the volume of the fermentation tank.

[0010] The fermentation raw materials include the following components in volume percentage (based on the volume of the fermenter): 40-60% fermentation medium, 0.1-1.2% defoamer, 10%-20% Ganoderma lucidum seed liquid, and water is added to make up to 60-80% of the volume of the fermenter.

[0011] Preferably, the fermentation raw materials account for 70% of the volume of the fermentation tank, and the fermentation raw materials include the following components in volume percentage: 44% fermentation medium, 1% defoaming agent, 20% Ganoderma lucidum strain seed liquid, and water is added to make up to 70% of the volume of the fermentation tank.

[0012] The fermentation medium has the following formula: carbon source 10-200 g / L, nitrogen source 1-20 g / L, magnesium ion 0.1-0.6 g / L, divalent iron ion 0.1-0.6 g / L, potassium ion 0.1-0.6 g / L, sulfate ion 0.1-0.6 g / L, phosphate ion 0.1-0.6 g / L, and thiamine 0.01-0.1 g / L.

[0013] Specifically, the carbon source is any one or a combination of wine lees, cassava residue, potato residue, kelp residue, chili residue and traditional Chinese medicine residue; the nitrogen source is any one or a combination of amino acid fermentation waste liquid, corn steep liquor, peptone and yeast extract powder.

[0014] The aforementioned distiller's grains, cassava residue, potato residue, kelp residue, chili residue, and traditional Chinese medicine residue must be ground and passed through a 40-mesh sieve before use. The distiller's grains were provided by Luzhou Laojiao; the cassava residue was provided by Guangxi Liyuan Group; and the potato residue, kelp residue, chili residue, and traditional Chinese medicine residue were purchased from Zhide Chemical (Shandong) Group Co., Ltd.

[0015] Preferably, the carbon source is a combination of cassava residue and potato residue; more preferably, the carbon source is a combination of cassava residue and potato residue in a weight ratio of 1-9:1-9; most preferably, the carbon source is a combination of cassava residue and potato residue in a weight ratio of 3:7, and the added amount is 30g / L cassava residue + 70g / L potato residue.

[0016] Preferably, the nitrogen source is corn steep liquor, and the addition amount is 14 g / L.

[0017] Preferably, the fermentation medium has a formula of: 30g / L+70g / L cassava residue+potato residue, 14g / L corn steep liquor, 0.4g / L MgSO4, 0.1g / L FeSO4·7H2O, 0.1g / L KH2PO4, and 0.01g / L VB1.

[0018] The Ganoderma lucidum includes but is not limited to any one or a combination of the red Ganoderma lucidum and the red Ganoderma lucidum described in the examples. In the prior art, any strain belonging to the Polyporaceae family can be used as the fermentation strain of the present invention.

[0019] Preferably, the Ganoderma lucidum strain is Ganoderma lucidum.

[0020] The above-mentioned red ganoderma and red ganoderma are numbered GXGD-3 and GXGD-11 in our company's culture library, and were purchased from Tianda Edible Fungi Research Institute and Guangzhou Longdong Edible Fungi Culture Factory respectively.

[0021] A method for preparing fermented feed is also within the scope of protection of the present invention, comprising the following steps:

[0022] (1) inoculating the mycelium of a Ganoderma lucidum strain into a seed liquid culture medium to obtain a Ganoderma lucidum strain seed liquid;

[0023] (2) inoculating the Ganoderma lucidum strain seed liquid obtained in step (1) into a fermentation tank for fermentation and culturing to obtain a fermentation product;

[0024] (3) The fermentation product obtained in step (2) is centrifuged, and the precipitate is dried to obtain the fermented feed.

[0025] Wherein, in step (1), the size of the fungus skin is 6*6mm, and 4 pieces are inoculated.

[0026] Wherein, in step (1), the seed liquid culture medium comprises: 10-30 g / L carbon source, 5-10 g / L amino acid fermentation waste liquid, 0.1-0.6 g / L MgSO4, 0.1-0.6 g / L FeSO4·7H2O, 0.1-0.6 g / L KH2PO4, and 0.01-0.1 g / L VB1; specifically, the carbon source comprises cassava residue and / or potato residue.

[0027] Preferably, the seed liquid culture medium is: 20g / L potato residue, 5g / L amino acid fermentation waste liquid, 0.4g / L MgSO4, 0.1g / L FeSO4·7H2O, 0.1g / L KH2PO4, and 0.01g / L VB1.

[0028] Wherein, in step (1), the culture conditions are: 23-27° C., 100-200 rpm, and culturing for 2-8 days.

[0029] Preferably, the culture conditions are: 25° C., 170 rpm, for 4 days.

[0030] Wherein, in step (2), the amount of the Ganoderma lucidum seed solution added is 10%-22% v / v, and the preferred inoculation amount is 20% v / v.

[0031] In step (2), the fermentation culture is carried out under the following conditions: pH 4-6, rotation speed 50-300 rpm, ventilation volume 0.8-1.6 vvm, and culture at 23-27°C for 2-8 days. Preferably, the fermentation conditions are: pH 5.0, rotation speed 100 rpm, ventilation volume 1.0 vvm, and culture at 25°C for 4 days.

[0032] The application of the fermented feed in broiler feeding is also within the scope of the present application.

[0033] Beneficial effects:

[0034] (1) The present application screens substrates suitable for the growth of Ganoderma lucidum GXGD-3 through different by-products flat plate.

[0035] (2) The present application uses by-products raw materials such as distiller's grains, cassava residues, potato residues, kelp residues, pepper residues and traditional Chinese medicine residues as substrates, screens fermentation strains, optimizes fermentation process and fermentation conditions, and uses liquid fermentation technology to prepare a functional feed with low fermentation cost and rich in nutritional components, so as to realize the purpose of replacing soybean meal as animal feed.

[0036] (3) The present application uses biological technology to process unconventional feed, and expands the utilization of unconventional feed resources.

[0037] (4) The present application uses Ganoderma lucidum as a fermentation strain, and the product after fermentation contains Ganoderma lucidum polysaccharide, which can effectively protect the animal intestinal tract and maintain the health of the animal intestinal tract. BRIEF DESCRIPTION OF DRAWINGS

[0038] The above and / or other aspects of the present application will become apparent by referring to the following description and the accompanying drawings.

[0039] Figure 1 Figure for the growth state of GXGD-3 and GXGD-11 strains on different by-product raw material solid culture medium. DETAILED DESCRIPTION

[0040] 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.

[0041] Example 1: Screening of Ganoderma lucidum fermentation substrates

[0042] After activation, two Ganoderma lucidum strains GXGD-3 (red Ganoderma lucidum, purchased from Tianda Edible Fungus Research Institute) and GXGD-11 (red Ganoderma lucidum, purchased from Guangzhou Longdong Edible Fungus Seed Factory) were inoculated in solid culture medium with six kinds of by-product raw materials (distiller's grains provided by Luzhou Laojiao; cassava residues provided by Guangxi Lixuan Group; potato residues, kelp residues, pepper residues and traditional Chinese medicine residues purchased from Tode Chemical (Shandong) Group Co., Ltd.) as the only carbon and nitrogen source at an inoculation amount of 20% v / v, and cultured at 25℃ for 7 days. The mycelial growth rate and growth state of the two strains were calculated.

[0043] The formula of the solid culture medium of different by-product raw materials is: 3% by-product raw materials, 2.5% agar, and all by-product raw materials are crushed and passed through a 40-mesh sieve before use.

[0044] The experimental results are as follows Figure 1 As shown in Table 1. Figure 1 As can be seen from Table 1, the growth rates of GXGD-3 and GXGD-11 in the cassava residue and potato residue plates are faster, among which the growth performance of GXGD-3 is more outstanding than that of GXGD-11, and the growth rate in the cassava residue and potato residue plates is the fastest, both at 1.16 cm / d. Figure 1 It can also be seen that the mycelium on the cassava residue and potato residue plates is evenly distributed, thick, and the mycelium growth state is better than that on the other four substrate plates. This shows that GXGD-3 is more suitable for growing in cassava residue and potato residue. (Note: Figure 1 The ef-5 marked on the plates of different byproduct raw materials is GXGD-3, and the ef-6 is GXGD-11. ef-5 and ef-6 are the original numbers of the red Ganoderma and red Ganoderma strains in the company's strain library. Due to the reorganization of the company's strain library, they were changed to the current numbers GXGD-3 and GXGD-11 respectively.

[0045] Table 1 Growth rate of Ganoderma lucidum on different raw materials

[0046] Full name strain potato scraps Cassava residue lees Kelp residue Chinese medicine residue Chili residue Red Ganoderma GXGD-3 1.16 1.16 0.96 0.51 0.73 0.23 Chizhi GXGD-11 1.11 1.09 0.91 0.47 0.69 0.31

[0047] Note: Mycelial growth rate (cm / d), formula = growth rate = diameter / days.

[0048] Crude protein, crude fiber (cellulose, hemicellulose, lignin), and ash were used as test indicators to investigate the compositional analysis of various by-product raw materials. Crude protein was determined according to GB / T6432-2018, "Determination of crude protein in feeds—Kjeldahl method," crude fiber according to GB / T6434-2006, "Determination of crude fiber in feeds—Filtration method," and ash according to GB / T5009.4-2006, "Determination of ash in foods." The results are shown in Table 2.

[0049] Table 2 Component analysis of 6 different by-product raw materials

[0050]

[0051] Combining the data in Table 1 and Table 2, we can see that GXGD-3 and GXGD-11 are more likely to utilize raw materials with high cellulose and hemicellulose content, such as potato residue and cassava residue. Although wine lees also contain 41.93% cellulose, its lignin content is too high and is not easily utilized by GXGD-3 and GXGD-11. Figure 1 The growth of GXGD-3 and GXGD-11 in 6 raw material plates was studied, and it was determined that Ganoderma lucidum (GXGD-3) was the target Ganoderma lucidum strain, and potato residue and cassava residue were the Ganoderma lucidum fermentation substrates.

[0052] Example 2 Optimization of Ganoderma lucidum seed liquid culture medium

[0053] A seed culture medium containing different inducers was prepared using 5 g / L of amino acid fermentation wastewater, 0.4 g / L of MgSO₄, 0.1 g / L of FeSO₄·7H₂O, 0.1 g / L of KH₂PO₄, and 0.01 g / L of VB1 as the basal medium. Additional inducers included 10 g / L, 20 g / L, and 30 g / L of cassava residue; 10 g / L, 20 g / L, and 30 g / L of potato residue; or 5 g / L, 10 g / L, and 15 g / L of cassava and potato residue. The activated GXGD-3 strain was inoculated into these different seed culture media and cultured at 25°C and 170 rpm for approximately 4 days to obtain a seed culture rich in GXGD-3 mycelium. The seed culture was centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected and dried at 65°C. Crude protein was used as an indicator (Table 3). Testing was performed according to the method specified in GB / T6432-2018, "Determination of Crude Protein in Feeds - Kjeldahl Method." Initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein increase rate, and overall total protein increase rate were calculated to identify the optimal inducer type and addition amount. Crude protein increase rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein; overall total protein increase rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein; total protein (g / L) = crude protein * product dry weight / 100.

[0054] Table 3 Growth of GXGD-3 under 10 different combinations of inducers

[0055]

[0056] As shown in Table 3, the group supplemented with 20 g / L of potato residue achieved the highest crude protein content at 35.05% after 4 days of culture, with a total protein content of 13.46 g / L. This represents a 103.19% increase in crude protein and a 138.63% increase in total protein compared to the initial fermentation period. Therefore, 20 g / L of potato residue is the optimal inducer. The optimized Ganoderma lucidum seed culture medium consists of 20 g / L of potato residue, 5 g / L of amino acid fermentation wastewater, 0.4 g / L of MgSO₄, 0.1 g / L of FeSO₄·7H₂O, 0.1 g / L of KH₂PO₄, and 0.01 g / L of VB1.

[0057] Example 3 Optimization of Ganoderma lucidum fermentation medium

[0058] The basal medium was composed of 5 g / L amino acid fermentation waste liquid, 0.4 g / L MgSO4, 0.1 g / L FeSO4·7H2O, 0.1 g / L KH2PO4, and 0.01 g / L VB1. Different fermentation media were composed of 50 g / L, 100 g / L, 150 g / L, and 200 g / L cassava residue or 50 g / L, 100 g / L, 150 g / L, and 200 g / L potato residue or 25 g / L+25 g / L, 50 g / L+50 g / L, 75 g / L+75 g / L, and 100 g / L+100 g / L cassava residue + potato residue, respectively. The seed solution rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated into different fermentation media at an inoculum size of 20% v / v and cultured at 25° C. and 170 rpm for about 4 days. After the fermentation product is cultivated, the precipitate is collected after centrifugation at 4000 rpm for 20 minutes, dried at 65 ° C, and crude protein is used as the detection index (Table 4). The detection is carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed - Kjeldahl nitrogen determination method". Calculate the initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein improvement rate and overall total protein improvement rate and other indicators to screen out the best raw material addition combination in the fermentation medium. Crude protein improvement rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein; overall total protein improvement rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein; total protein (g / L) = crude protein * product dry weight / 100.

[0059] Table 4 Growth of GXGD-3 under 13 different raw material combinations

[0060]

[0061] As shown in Table 4, the crude protein content of the formula with 50g / L cassava residue and 50g / L potato residue is 38.38%, and the total protein is 9.15g / L. This is a 117.91% increase in crude protein and a 90.94% increase in total protein compared to the initial fermentation level. Therefore, when the total addition amount is 100g / L, 50g / L cassava residue and 50g / L potato residue is the optimal addition amount. On this basis, the ratio of cassava residue to potato residue can be further optimized.

[0062] The basal medium was composed of 5 g / L amino acid fermentation waste liquid, 0.4 g / L MgSO4, 0.1 g / L FeSO4·7H2O, 0.1 g / L KH2PO4, and 0.01 g / L VB1. 10 g / L+90 g / L, 20 g / L+80 g / L, 30 g / L+70 g / L, 40 g / L+60 g / L, 50 g / L+50 g / L, 60 g / L+40 g / L, 70 g / L+30 g / L, 80 g / L+20 g / L, and 90 g / L+10 g / L of cassava residue and potato residue were added, respectively, to form different fermentation media. The seed solution rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated into different fermentation media at an inoculum size of 20% v / v and cultured at 25 ° C and 170 rpm for about 4 days. After the fermentation product was cultivated, the precipitate was collected after centrifugation at 4000 rpm for 20 minutes, dried at 65 ° C, and crude protein was used as the detection index (Table 5). The test was carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed - Kjeldahl nitrogen determination method". The initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein improvement rate and overall total protein improvement rate were calculated to screen out the best combination of cassava residue and potato residue in the fermentation medium. Crude protein improvement rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein; overall total protein improvement rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein; total protein (g / L) = crude protein * product dry weight / 100.

[0063] Table 5 Growth of GXGD-3 under 9 different combinations of cassava residue and potato residue

[0064]

[0065] As shown in Table 5, the crude protein content of the fermentation medium containing 30 g / L + 70 g / L of cassava residue and potato residue was 38.95%, and the total protein content was 9.24 g / L. This represents a 127.64% increase in crude protein and a 110.26% increase in total protein compared to the initial fermentation period. Therefore, the 30 g / L + 70 g / L cassava residue + potato residue formula is the optimal addition amount to the fermentation medium.

[0066] Example 4 Optimization of the type and amount of organic nitrogen source added to the fermentation medium

[0067] (1) Category optimization

[0068] The type of nitrogen source has a great influence on the fermentation intensity. Four commonly used organic nitrogen source raw materials were selected, and the content of organic nitrogen was calculated to ensure that the nitrogen source content in the formula was consistent. Based on the fermentation medium optimized in Example 3, the type of organic nitrogen source in the fermentation medium was optimized. The fermentation medium formula was as follows: 30g / L+70g / L cassava residue+potato residue, 5g / L amino acid fermentation waste liquid (or 16g / L corn steep liquor or 6.4g / L peptone or 6.4g / L yeast extract), 0.4g / L MgSO4, 0.1g / L FeSO4·7H2O, 0.1g / L KH2PO4, 0.1g / L VB1, and the seed liquid rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated into the above-mentioned fermentation medium containing different nitrogen sources at an inoculum amount of 20% v / v, and cultured at 25°C and 170rpm for about 4 days. After the fermentation product is cultivated, it is centrifuged at 4000 rpm for 20 minutes, and the precipitate is collected and dried at 65°C. The nitrogen content of the four nitrogen sources and crude protein (Table 6) are used as test indicators. The test is carried out according to the method in GB / T6432-2018 "Determination of crude protein in feeds - Kjeldahl method". The initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein improvement rate and overall total protein improvement rate are calculated to screen out the best inorganic nitrogen and total nitrogen types. Crude protein improvement rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein; overall total protein improvement rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein; total protein (g / L) = crude protein * product dry weight / 100.

[0069] Among them, amino acid fermentation waste liquid was provided by Ningxia Yipin Biotechnology Co., Ltd.; corn steep liquor was provided by COFCO Biotechnology Co., Ltd.; and peptone and yeast extract powder were purchased from Angel Yeast Co., Ltd.

[0070] Table 6 Growth of GXGD-3 under four different organic nitrogen sources

[0071]

[0072] The total nitrogen contents of the four nitrogen sources, amino acid fermentation waste liquid, corn steep liquor, peptone and yeast extract powder, were 3.11%, 6.93%, 7.15% and 8.72%, respectively. As can be seen from Table 6, the crude protein content of the formula with the addition of 16 g / L corn steep liquor was the highest, reaching 40.50%, and the total protein was 9.74 g / L, which was increased by 137.96% compared with the initial crude protein and increased by 138.73% compared with the initial total protein. Therefore, 16 g / L corn steep liquor was the best type of organic nitrogen source, and the addition amount of corn steep liquor was optimized based on this.

[0073] (2) Addition amount optimization

[0074] The addition amount of corn steep liquor in the fermentation medium was optimized, and the fermentation medium formula was as follows: 30 g / L + 70 g / L cassava dregs + potato dregs, corn steep liquor 12 g / L (or 14 g / L, 16 g / L, 18 g / L, 20 g / L), MgSO4 0.4 g / L, FeSO4·7H2O 0.1 g / L, KH2PO4 0.1 g / L, VB1 0.01 g / L. The seed liquid rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated into the above fermentation medium containing different addition amounts of corn steep liquor at an inoculation amount of 20% v / v, and cultured at 25°C and 170 rpm for about 4 days. After the fermentation was completed, the fermentation product was centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected and dried at 65°C. The crude protein was used as the detection index (Table 7). The detection was carried out according to the method in GB / T6432-2018 “Determination of crude protein in feed by Kjeldahl method”. The initial crude protein, initial total protein, crude protein, total protein, crude protein increase rate and total protein increase rate were calculated, and the best addition amount of corn steep liquor was selected. Crude protein increase rate (%) = 100 * (total protein - initial crude protein) / initial crude protein; total protein increase rate (%) = 100 * (total protein - initial total protein) / initial total protein; total protein (g / L) = crude protein * product dry weight / 100.

[0075] Table 7: Growth of GXGD-3 under 5 different addition amounts of corn steep liquor

[0076]

[0077]

[0078] The results are shown in Table 7. The group supplemented with 16 g / L corn steep liquor had the highest crude protein content of 40.99% and a total protein content of 9.61 g / L, representing a 133.43% increase in crude protein and a 133.82% increase in total protein compared to the initial fermentation stage. The group supplemented with 14 g / L corn steep liquor had a crude protein content of 40.40% and a total protein content of 9.53 g / L, representing a 140.33% increase in crude protein and a 144.36% increase in total protein compared to the initial fermentation stage. Considering the protein increase rate, total protein content, and fermentation cost, 14 g / L corn steep liquor was selected as the optimal addition. The fermentation medium formulation at this time was: 30 g / L + 70 g / L cassava and potato residues, 14 g / L corn steep liquor, 0.4 g / L MgSO₄, 0.1 g / L FeSO₄·7H₂O, 0.1 g / L KH₂PO₄, and 0.01 g / L VB1.

[0079] Example 5 Single factor experiment to optimize fermentation conditions

[0080] The fermentation conditions were optimized in a 10 L fermenter using a single factor experiment.

[0081] (1) Optimization of inoculum size

[0082] The seed solution rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated into a 10L fermenter containing the fermentation medium optimized in Example 4 at an inoculum size of 10%, 15%, or 20% v / v. The liquid volume was 70%, the fermentation medium was fixed to 5L, 1% defoamer was added, and the fermenter was sterilized with steam at 121°C for 30 minutes. After sterilization, 1-2L of sterile steam condensate was added to the fermenter, with an initial pH of 5.0. The liquid level was adjusted to 7L with sterile water in the water bottle, the speed was 100rpm, the ventilation volume was 0.8vvm, and the culture was carried out at 25°C for about 4 days. The fermentation product after the culture was centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected and dried at 65°C. Crude protein was used as the detection index. Detection was carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed - Kjeldahl nitrogen method". Calculate the initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein improvement rate and overall total protein improvement rate to screen out the optimal inoculation amount.

[0083] Wherein, the fermentor tank is connected with a 1L acid feed bottle, a 1L alkali feed bottle (acid and alkali feed bottles are used to regulate pH) and a 5L water replenishment bottle (the water replenishment bottle is used to complete the fermentor tank liquid amount) at the feed interface. 500mL of 1% sulfuric acid is loaded into the 1L acid feed bottle, 500mL of ammoniacal liquor is loaded into the 1L alkali feed bottle, and 3L of water is loaded into the 5L water replenishment bottle. Wherein, the acid feed bottle, the acid solution, the water replenishment bottle and the water all need to be bandaged in advance, sterilized at 121°C for 30 minutes in a high-pressure steam autoclave, and then inserted into the feed interface of the fermentor tank for use after sterilization.

[0084] The results showed (Table 8) that the crude protein content of the group with an inoculation amount of 20% v / v was 45.62%, and the overall total protein was 9.60 g / L, which was 129.82% higher than that at the beginning of fermentation, and the overall total protein was increased by 100.00%.

[0085] (2) Initial pH optimization

[0086] The seed solution rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated at an inoculum size of 20% v / v into a 10L fermenter containing the fermentation medium optimized in Example 4. The liquid volume was 70%, the fermentation medium was fixed to 5L, 1% defoamer was added, and the fermentation medium was sterilized with 121°C steam for 30 minutes. After sterilization, there was 1-2L of sterile steam condensate in the fermenter. The initial pH of 5.0 was adjusted to pH 4.0 or pH 6.0 with the acid and alkali solution in the feed bottle, and the liquid level was adjusted to 7L with sterile water in the water bottle. The speed was 100rpm, the ventilation volume was 0.8vvm, and the culture was carried out at 25°C for about 4 days. The fermentation product after the culture was centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected and dried at 65°C. Crude protein was used as the detection index. Detection was carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed - Kjeldahl method". Calculate initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein improvement rate, and overall total protein improvement rate to determine the optimal initial pH. Crude protein improvement rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein; overall total protein improvement rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein; Total protein (g / L) = crude protein * product dry weight / 100.

[0087] The results showed (Table 8) that the crude protein content of the group with an initial pH of 5.0 was 45.95%, and the overall total protein was 9.34 g / L, which was an increase of 134.44% in crude protein and 96.22% in overall total protein compared with the initial stage of fermentation.

[0088] (3) Speed ​​optimization

[0089] The seed liquid enriched in GXGD-3 mycelium obtained after optimization of Example 2 was inoculated into a 10L fermenter containing the optimized fermentation medium of Example 4 at an inoculation amount of 20% v / v, the liquid volume was 70%, the fermentation medium was adjusted to 5L, 1% antifoam was added, and the fermentation medium was sterilized at 121°C for 30 minutes. After sterilization, there was 1-2L of sterile steam condensate in the fermenter. The initial pH was 5.0, the liquid level was adjusted to 7L with sterile water in a water bottle, the rotation speed was 100rpm or 200rpm or 300rpm, the aeration amount was 0.8vvm, and the fermentation was carried out at 25°C for about 4 days. After the fermentation was completed, the fermentation product was centrifuged at 4000rpm for 20 minutes, the precipitate was collected, and the crude protein was detected as the detection index. The detection was carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed by Kjeldahl method". The crude protein, total protein, crude protein improvement rate and total protein improvement rate were calculated to select the best rotation speed. Crude protein improvement rate (%) = 100*(total protein-initial crude protein) / initial crude protein, total protein improvement rate (%) = 100*(total protein-initial total protein) / initial total protein, total protein (g / L) = crude protein*product dry weight / 100.

[0090] The results showed (Table 8) that the crude protein content of the fermentation rotation speed 100rpm group was 45.69%, the total protein was 9.62g / L, the crude protein was improved by 130.06% compared with the initial fermentation, and the total protein was improved by 100.42%.

[0091] (4) Aeration optimization

[0092] The seed solution rich in GXGD-3 mycelium obtained after optimization in Example 2 was inoculated at an inoculum size of 20% v / v into a 10L fermenter containing the fermentation medium optimized in Example 4. The liquid volume was 70%, the fermentation medium was fixed to 5L, 1% defoamer was added, and the fermenter was sterilized with steam at 121°C for 30 minutes. After sterilization, there was 1-2L of sterile steam condensate in the fermenter, the initial pH was 5.0, and the liquid level was adjusted to 7L with sterile water in the water bottle. The speed was 100rpm, the ventilation volume was 0.8vvm or 1.2vvm or 1.6vvm, and the culture was carried out at 25°C for about 4 days. The fermentation product after the culture was centrifuged at 4000 rpm for 20 minutes, and the precipitate was collected and dried at 65°C. Crude protein was used as the detection index. Detection was carried out according to the method in GB / T6432-2018 "Determination of crude protein in feed - Kjeldahl nitrogen method". Calculate the initial crude protein, initial overall total protein, crude protein, overall total protein, crude protein increase rate, and overall total protein increase rate to select the optimal ventilation rate. Crude protein increase rate (%) = 100 * (overall total protein - initial crude protein) / initial crude protein, overall total protein increase rate (%) = 100 * (overall total protein - initial overall total protein) / initial overall total protein, total protein (g / L) = crude protein * product dry weight / 100.

[0093] The results showed (Table 8) that the crude protein content of the ventilation rate 1.2 vvm group was 46.11%, and the overall total protein was 9.60 g / L, which was 132.29% higher than that in the initial fermentation stage, and the overall total protein was 100.00% higher.

[0094] Table 8 Growth of GXGD-3 under different conditions

[0095]

[0096] Based on the above results, subsequent orthogonal experiments were carried out in the vicinity of the above-mentioned single-factor optimal conditions to obtain the optimal fermentation condition combination.

[0097] Example 6 Orthogonal test to optimize fermentation conditions

[0098] In Example 5, single factor optimization was performed on the inoculum size, initial pH, rotation speed and ventilation rate in the fermentation conditions. The final optimal inoculum size was 20% v / v, initial pH 5.0, fermentation rotation speed 100 rpm and ventilation rate 1.2 vvm. Orthogonal experiment was carried out with single factor optimization conditions. L9(3 4A three-level, four-factor orthogonal experiment (Table 9) was conducted to analyze the inoculum size, initial pH, rotation speed, and ventilation rate in the fermentation formula. Cultures were maintained at 25°C for approximately four days. The fermentation product was centrifuged at 4000 rpm for 20 minutes, the precipitate collected, and dried at 65°C. Crude protein was used as the assay (Table 10). Determination was performed according to the method specified in GB / T 6432-2018, "Determination of Crude Protein in Feeds - Kjeldahl Method."

[0099] Table 9: Orthogonal test factors and levels

[0100]

[0101] Table 10 Orthogonal experiment range analysis

[0102]

[0103] Analysis of the orthogonal experiment results in Table 10 shows that the factors influencing the experiment are C > B > A > D, and the optimal combination is C2B2A2D1, which is defined as: inoculum size 20% v / v, initial pH 5.0, rotation speed 100 rpm, and ventilation rate 1.0 vvm. The crude protein content after fermentation was 47.83%.

[0104] Example 7 Verification of the optimal fermentation medium and fermentation conditions

[0105] The clean bench was disinfected with ultraviolet light for 30 minutes, and four 6*6 mm fungus skins were printed from the GXGD-3 seed plate using an Oxford cup in the clean bench. The fungus skins were added to the seed liquid culture medium optimized in Example 2 (20 g / L potato residue, 5 g / L amino acid fermentation waste liquid, 0.4 g / L MgSO4, 0.1 g / L FeSO4·7H2O, 0.1 g / L KH2PO4, 0.1 g / L VB1), and cultured at 25 ° C and 170 rpm for about 4 days.

[0106] The optimal fermentation conditions were verified using a 50L fermentor tank with a liquid capacity of 70%. A total of 22L of fermentation medium (30g / L+70g / L cassava residues and potato residues, 14g / L corn steep liquor, 0.4g / L MgSO4, 0.1g / L FeSO4·7H2O, 0.1g / L KH2PO4, and 0.01g / L VB1) was added, followed by 1% defoamer and sterilization at 121°C for 30 minutes. After sterilization, 1-2L of sterile steam condensed water was added to the fermentor tank, followed by 20% v / v seed solution. The initial pH was adjusted to 5.0 using the acid and alkali in the feed bottle, and the liquid level was adjusted to 35L using the sterile water in the feed bottle. The fermentor tank was maintained at a ventilation rate of 1.0 vvm and a rotational speed of 100 rpm for 4 days. The crude protein content after fermentation was 48.00%.

[0107] Crude protein, crude fiber, and ash were used as test indicators for component analysis of the initial fermentation phase and the fermentation product. Crude protein was determined according to GB / T6432-2018, "Determination of crude protein in feeds - Kjeldahl method," crude fiber according to GB / T6434-2006, "Determination of crude fiber in feeds - filtration method," and ash according to GB / T5009.4-2006, "Determination of ash in foods."

[0108] Test data (Table 11) showed that the crude protein content of the fermented product increased significantly, by 165.04%, while the cellulose, hemicellulose, lignin, and ash contents decreased significantly, by 81.68%, 62.76%, 64.64%, and 52.63%, respectively. This suggests that the residual starch and crude fiber in the cassava and potato residues can be utilized by Hongzhi GXGD-3 and converted into its own bacterial protein, thereby increasing the protein content.

[0109] Table 11 Component analysis of early fermentation and fermentation products

[0110]

[0111] Example 8 Broiler Feeding Test

[0112] The fermented product obtained in Example 7 was used as Ganoderma feed for broiler chickens. 150 one-day-old broilers were randomly divided into three treatment groups (control group, 5% Ganoderma feed group, and 10% Ganoderma feed group) according to body weight, with 5 replicates per treatment group and 10 chickens per replicate.

[0113] The experimental period lasted for 6 weeks. The control group was fed a basal diet, while the 5% and 10% Ganoderma feed groups each replaced part of the soybean meal with equal crude protein content (Table 12). The basal diet followed the NRC Broiler Feeding Standards. Broilers had free access to feed and water during the feeding period. Daily hygiene and epidemic prevention procedures were followed as normal during the experimental period. Feed intake was recorded daily, and average feed intake, average daily gain, and feed-to-gain ratio were calculated.

[0114] The test results, shown in Table 13, show improvements in average daily gain, average feed intake, and feed-to-gain ratio compared to the control group, indicating that 5-10% of Ganoderma lucidum feed can replace soybean meal in feed. Furthermore, Ganoderma lucidum is rich in polysaccharides, which have anti-inflammatory, immune-regulating, and antioxidant properties. The polysaccharides in fermented feed can effectively protect the broiler chickens' intestines and maintain intestinal health. Therefore, Ganoderma lucidum feed is a natural, active "health supplement."

[0115] Table 12 Feed comparison table

[0116]

[0117] Table 13 Effects of different levels of Ganoderma feed on growth performance of broilers

[0118]

[0119]

[0120] The present invention provides a fermented feed prepared using Ganoderma lucidum, and its preparation and application methods and concepts. There are many specific methods and approaches for implementing this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A fermented feed prepared using Ganoderma lucidum, characterized in that: The fermented feed is prepared by fermenting fermentation raw materials; Wherein, the fermentation raw materials account for 60-80% of the volume of the fermentation tank; The fermentation raw materials include the following components in volume percentage: 40-60% fermentation medium, 0.1-1.2% defoamer, 20% Ganoderma lucidum seed liquid, and water is added to make up to 60-80% of the volume of the fermentation tank; The fermentation medium has the following formula: carbon source 100 g / L, nitrogen source 14 g / L, magnesium ion 0.1-0.6 g / L, divalent iron ion 0.1-0.6 g / L, potassium ion 0.1-0.6 g / L, sulfate ion 0.1-0.6 g / L, phosphate ion 0.1-0.6 g / L, thiamine 0.01-0.1 g / L; Wherein, the Ganoderma lucidum is Ganoderma lucidum; the carbon source is cassava residue and potato residue, and the cassava residue and potato residue are combined in a weight ratio of 3:7; the nitrogen source is corn steep liquor; The fermentation preparation has the following fermentation conditions: initial pH 5.0, rotation speed 100 rpm, ventilation volume 1.0 vvm, and culture for 4 days.

2. The fermented feed according to claim 1, characterized in that The fermentation raw materials account for 70% of the volume of the fermentation tank, and the fermentation raw materials include the following components in volume percentage: 44% fermentation medium, 1% defoaming agent, 20% Ganoderma lucidum seed liquid, and water is added to make up to 70% of the volume of the fermentation tank.

3. The method for preparing fermented feed according to claim 1, characterized in that: The steps include: (1) inoculating the skin of Ganoderma lucidum into a seed liquid culture medium to obtain Ganoderma lucidum seed liquid; (2) adding the fermentation medium, defoaming agent, and the Ganoderma lucidum seed solution obtained in step (1) into a fermentation tank, adding water to make up to 60-80% of the volume of the fermentation tank, and performing fermentation culture to obtain a fermentation product; (3) The fermentation product obtained in step (2) is centrifuged, and the precipitate is dried to obtain the fermented feed.

4. The preparation method according to claim 3, characterized in that In step (1), the seed liquid culture medium comprises: 10-30 g / L carbon source, 5-10 g / L amino acid fermentation waste liquid, 0.1-0.6 g / L magnesium ion, 0.1-0.6 g / L divalent iron ion, 0.1-0.6 g / L potassium ion, 0.1-0.6 g / L sulfate ion, 0.1-0.6 g / L phosphate ion, and 0.01-0.1 g / L thiamine.

5. The preparation method according to claim 4, characterized in that The carbon source of the seed liquid culture medium is cassava residue and / or potato residue.

6. The preparation method according to claim 3, characterized in that In step (1), the culture conditions are: 23-27°C, 100-200 rpm, 2-8 days.

7. Use of the fermented feed according to any one of claims 1 to 2 in feeding broiler chickens.

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